{"database": "metadata", "table": "run_metadata", "is_view": false, "human_description_en": "where devstage_curation_coarse = \"Embryo\" and technology = \"indrops\"", "rows": [[32725, "SRR29398864", "SRX24912671", "SRS21618605", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 2 3", "GSM8327220", null, "source name:whole embryo|tissue:whole embryo|treatment:control and emi1 homozygous mutants|geo loc name:missing|collection date:missing", "Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 2 3", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:control and emi1 homozygous mutants", "GSM8327220", "GSM8327220: Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 2 3; Danio rerio; OTHER", "GSM8327220 r1", "GSM8327220", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "pert_exp3_brep1_2_3_trep2_3_S0_L001_R1_001.fastq.gz pert_exp3_brep1_2_3_trep2_3_S0_L001_R2_001.fastq.gz pert_exp3_brep1_2_3_trep2_3_S0_L001_R3_001.fastq.gz pert_exp3_brep1_2_3_trep2_3_S0_L001_R4_001.fastq.gz", "fastq fastq fastq fastq", 10196913181.0, 112053991.0, "GSM8327220 r1", "0:61 1:8 2:8 3:14", "A:1907833453;C:1411947502;G:1388620349;T:2126120065;N:772082", 61, 8, 8, 14, 1907833453, 1411947502, 1388620349, 2126120065, 772082, "SRX24912671", "SRS21618605", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32726, "SRR29398865", "SRX24912671", "SRS21618605", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 2 3", "GSM8327220", null, "source name:whole embryo|tissue:whole embryo|treatment:control and emi1 homozygous mutants|geo loc name:missing|collection date:missing", "Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 2 3", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:control and emi1 homozygous mutants", "GSM8327220", "GSM8327220: Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 2 3; Danio rerio; OTHER", "GSM8327220 r1", "GSM8327220", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "pert_exp3_brep1_2_3_trep2_3_S0_L002_R1_001.fastq.gz pert_exp3_brep1_2_3_trep2_3_S0_L002_R2_001.fastq.gz pert_exp3_brep1_2_3_trep2_3_S0_L002_R3_001.fastq.gz pert_exp3_brep1_2_3_trep2_3_S0_L002_R4_001.fastq.gz", "fastq fastq fastq fastq", 10073565229.0, 110698519.0, "GSM8327220 r2", "0:61 1:8 2:8 3:14", "A:1882668771;C:1393477449;G:1377270278;T:2098291783;N:901378", 61, 8, 8, 14, 1882668771, 1393477449, 1377270278, 2098291783, 901378, "SRX24912671", "SRS21618605", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32727, "SRR29398866", "SRX24912671", "SRS21618605", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 2 3", "GSM8327220", null, "source name:whole embryo|tissue:whole embryo|treatment:control and emi1 homozygous mutants|geo loc name:missing|collection date:missing", "Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 2 3", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:control and emi1 homozygous mutants", "GSM8327220", "GSM8327220: Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 2 3; Danio rerio; OTHER", "GSM8327220 r1", "GSM8327220", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "pert_exp3_brep1_2_3_trep2_3_S0_L003_R1_001.fastq.gz pert_exp3_brep1_2_3_trep2_3_S0_L003_R2_001.fastq.gz pert_exp3_brep1_2_3_trep2_3_S0_L003_R3_001.fastq.gz pert_exp3_brep1_2_3_trep2_3_S0_L003_R4_001.fastq.gz", "fastq fastq fastq fastq", 10334862265.0, 113569915.0, "GSM8327220 r3", "0:61 1:8 2:8 3:14", "A:1932620835;C:1429967805;G:1408958497;T:2155416872;N:800806", 61, 8, 8, 14, 1932620835, 1429967805, 1408958497, 2155416872, 800806, "SRX24912671", "SRS21618605", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32728, "SRR29398867", "SRX24912671", "SRS21618605", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 2 3", "GSM8327220", null, "source name:whole embryo|tissue:whole embryo|treatment:control and emi1 homozygous mutants|geo loc name:missing|collection date:missing", "Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 2 3", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:control and emi1 homozygous mutants", "GSM8327220", "GSM8327220: Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 2 3; Danio rerio; OTHER", "GSM8327220 r1", "GSM8327220", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "pert_exp3_brep1_2_3_trep2_3_S0_L004_R1_001.fastq.gz pert_exp3_brep1_2_3_trep2_3_S0_L004_R2_001.fastq.gz pert_exp3_brep1_2_3_trep2_3_S0_L004_R3_001.fastq.gz pert_exp3_brep1_2_3_trep2_3_S0_L004_R4_001.fastq.gz", "fastq fastq fastq fastq", 10120552533.0, 111214863.0, "GSM8327220 r4", "0:61 1:8 2:8 3:14", "A:1891306008;C:1401185707;G:1383554545;T:2107193563;N:866820", 61, 8, 8, 14, 1891306008, 1401185707, 1383554545, 2107193563, 866820, "SRX24912671", "SRS21618605", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32729, "SRR29398868", "SRX24912670", "SRS21618604", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 1", "GSM8327219", null, "source name:whole embryo|tissue:whole embryo|treatment:control and emi1 homozygous mutants|geo loc name:missing|collection date:missing", "Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 1", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:control and emi1 homozygous mutants", "GSM8327219", "GSM8327219: Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 1; Danio rerio; OTHER", "GSM8327219 r1", "GSM8327219", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "pert_exp3_brep1_2_3_trep1_S0_L001_R1_001.fastq.gz pert_exp3_brep1_2_3_trep1_S0_L001_R2_001.fastq.gz pert_exp3_brep1_2_3_trep1_S0_L001_R3_001.fastq.gz pert_exp3_brep1_2_3_trep1_S0_L001_R4_001.fastq.gz", "fastq fastq fastq fastq", 11110219029.0, 122090319.0, "GSM8327219 r1", "0:61 1:8 2:8 3:14", "A:2089393711;C:1503585110;G:1499880828;T:2353645774;N:1004036", 61, 8, 8, 14, 2089393711, 1503585110, 1499880828, 2353645774, 1004036, "SRX24912670", "SRS21618604", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32730, "SRR29398869", "SRX24912670", "SRS21618604", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 1", "GSM8327219", null, "source name:whole embryo|tissue:whole embryo|treatment:control and emi1 homozygous mutants|geo loc name:missing|collection date:missing", "Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 1", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:control and emi1 homozygous mutants", "GSM8327219", "GSM8327219: Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 1; Danio rerio; OTHER", "GSM8327219 r1", "GSM8327219", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "pert_exp3_brep1_2_3_trep1_S0_L002_R1_001.fastq.gz pert_exp3_brep1_2_3_trep1_S0_L002_R2_001.fastq.gz pert_exp3_brep1_2_3_trep1_S0_L002_R3_001.fastq.gz pert_exp3_brep1_2_3_trep1_S0_L002_R4_001.fastq.gz", "fastq fastq fastq fastq", 10960533857.0, 120445427.0, "GSM8327219 r2", "0:61 1:8 2:8 3:14", "A:2060454932;C:1484110501;G:1481072325;T:2320147865;N:1385424", 61, 8, 8, 14, 2060454932, 1484110501, 1481072325, 2320147865, 1385424, "SRX24912670", "SRS21618604", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32731, "SRR29398870", "SRX24912670", "SRS21618604", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 1", "GSM8327219", null, "source name:whole embryo|tissue:whole embryo|treatment:control and emi1 homozygous mutants|geo loc name:missing|collection date:missing", "Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 1", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:control and emi1 homozygous mutants", "GSM8327219", "GSM8327219: Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 1; Danio rerio; OTHER", "GSM8327219 r1", "GSM8327219", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "pert_exp3_brep1_2_3_trep1_S0_L003_R1_001.fastq.gz pert_exp3_brep1_2_3_trep1_S0_L003_R2_001.fastq.gz pert_exp3_brep1_2_3_trep1_S0_L003_R3_001.fastq.gz pert_exp3_brep1_2_3_trep1_S0_L003_R4_001.fastq.gz", "fastq fastq fastq fastq", 11164280309.0, 122684399.0, "GSM8327219 r3", "0:61 1:8 2:8 3:14", "A:2098548238;C:1513159748;G:1508645874;T:2362720749;N:673730", 61, 8, 8, 14, 2098548238, 1513159748, 1508645874, 2362720749, 673730, "SRX24912670", "SRS21618604", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32732, "SRR29398871", "SRX24912670", "SRS21618604", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 1", "GSM8327219", null, "source name:whole embryo|tissue:whole embryo|treatment:control and emi1 homozygous mutants|geo loc name:missing|collection date:missing", "Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 1", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:control and emi1 homozygous mutants", "GSM8327219", "GSM8327219: Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 1; Danio rerio; OTHER", "GSM8327219 r1", "GSM8327219", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "pert_exp3_brep1_2_3_trep1_S0_L004_R1_001.fastq.gz pert_exp3_brep1_2_3_trep1_S0_L004_R2_001.fastq.gz pert_exp3_brep1_2_3_trep1_S0_L004_R3_001.fastq.gz pert_exp3_brep1_2_3_trep1_S0_L004_R4_001.fastq.gz", "fastq fastq fastq fastq", 11112051587.0, 122110457.0, "GSM8327219 r4", "0:61 1:8 2:8 3:14", "A:2088203494;C:1504533428;G:1503021684;T:2351948933;N:1030338", 61, 8, 8, 14, 2088203494, 1504533428, 1503021684, 2351948933, 1030338, "SRX24912670", "SRS21618604", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32733, "SRR29398872", "SRX24912669", "SRS21618603", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Perturbation experiment 2 24 hpf biological replicate 3 technical replicate 1 to 4", "GSM8327218", null, "source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf|geo loc name:missing|collection date:missing", "Perturbation experiment 2 24 hpf biological replicate 3 technical replicate 1 to 4", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf", "GSM8327218", "GSM8327218: Perturbation experiment 2 24 hpf biological replicate 3 technical replicate 1 to 4; Danio rerio; OTHER", "GSM8327218 r1", "GSM8327218", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "pert_exp2_brep3_trep1_to_4_S0_L001_R1_001.fastq.gz pert_exp2_brep3_trep1_to_4_S0_L001_R2_001.fastq.gz pert_exp2_brep3_trep1_to_4_S0_L001_R3_001.fastq.gz pert_exp2_brep3_trep1_to_4_S0_L001_R4_001.fastq.gz", "fastq fastq fastq fastq", 10415939716.0, 114460876.0, "GSM8327218 r1", "0:61 1:8 2:8 3:14", "A:1937284334;C:1499506650;G:1405060643;T:2140026232;N:235577", 61, 8, 8, 14, 1937284334, 1499506650, 1405060643, 2140026232, 235577, "SRX24912669", "SRS21618603", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32734, "SRR29398873", "SRX24912669", "SRS21618603", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Perturbation experiment 2 24 hpf biological replicate 3 technical replicate 1 to 4", "GSM8327218", null, "source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf|geo loc name:missing|collection date:missing", "Perturbation experiment 2 24 hpf biological replicate 3 technical replicate 1 to 4", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf", "GSM8327218", "GSM8327218: Perturbation experiment 2 24 hpf biological replicate 3 technical replicate 1 to 4; Danio rerio; OTHER", "GSM8327218 r1", "GSM8327218", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "pert_exp2_brep3_trep1_to_4_S0_L002_R1_001.fastq.gz pert_exp2_brep3_trep1_to_4_S0_L002_R2_001.fastq.gz pert_exp2_brep3_trep1_to_4_S0_L002_R3_001.fastq.gz pert_exp2_brep3_trep1_to_4_S0_L002_R4_001.fastq.gz", "fastq fastq fastq fastq", 10347954344.0, 113713784.0, "GSM8327218 r2", "0:61 1:8 2:8 3:14", "A:1929648265;C:1483960768;G:1389914714;T:2132824805;N:192272", 61, 8, 8, 14, 1929648265, 1483960768, 1389914714, 2132824805, 192272, "SRX24912669", "SRS21618603", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32735, "SRR29398874", "SRX24912669", "SRS21618603", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Perturbation experiment 2 24 hpf biological replicate 3 technical replicate 1 to 4", "GSM8327218", null, "source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf|geo loc name:missing|collection date:missing", "Perturbation experiment 2 24 hpf biological replicate 3 technical replicate 1 to 4", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf", "GSM8327218", "GSM8327218: Perturbation experiment 2 24 hpf biological replicate 3 technical replicate 1 to 4; Danio rerio; OTHER", "GSM8327218 r1", "GSM8327218", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "pert_exp2_brep3_trep1_to_4_S0_L003_R1_001.fastq.gz pert_exp2_brep3_trep1_to_4_S0_L003_R2_001.fastq.gz pert_exp2_brep3_trep1_to_4_S0_L003_R3_001.fastq.gz pert_exp2_brep3_trep1_to_4_S0_L003_R4_001.fastq.gz", "fastq fastq fastq fastq", 10349635660.0, 113732260.0, "GSM8327218 r3", "0:61 1:8 2:8 3:14", "A:1930191842;C:1471248043;G:1399416562;T:2136531503;N:279910", 61, 8, 8, 14, 1930191842, 1471248043, 1399416562, 2136531503, 279910, "SRX24912669", "SRS21618603", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32736, "SRR29398875", "SRX24912669", "SRS21618603", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Perturbation experiment 2 24 hpf biological replicate 3 technical replicate 1 to 4", "GSM8327218", null, "source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf|geo loc name:missing|collection date:missing", "Perturbation experiment 2 24 hpf biological replicate 3 technical replicate 1 to 4", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf", "GSM8327218", "GSM8327218: Perturbation experiment 2 24 hpf biological replicate 3 technical replicate 1 to 4; Danio rerio; OTHER", "GSM8327218 r1", "GSM8327218", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "pert_exp2_brep3_trep1_to_4_S0_L004_R1_001.fastq.gz pert_exp2_brep3_trep1_to_4_S0_L004_R2_001.fastq.gz pert_exp2_brep3_trep1_to_4_S0_L004_R3_001.fastq.gz pert_exp2_brep3_trep1_to_4_S0_L004_R4_001.fastq.gz", "fastq fastq fastq fastq", 10428481518.0, 114598698.0, "GSM8327218 r4", "0:61 1:8 2:8 3:14", "A:1947321872;C:1488626558;G:1403484811;T:2150802963;N:284374", 61, 8, 8, 14, 1947321872, 1488626558, 1403484811, 2150802963, 284374, "SRX24912669", "SRS21618603", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32737, "SRR29398876", "SRX24912668", "SRS21618601", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Perturbation experiment 2 24 hpf biological replicate 2 technical replicate 1 to 6", "GSM8327217", null, "source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf|geo loc name:missing|collection date:missing", "Perturbation experiment 2 24 hpf biological replicate 2 technical replicate 1 to 6", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf", "GSM8327217", "GSM8327217: Perturbation experiment 2 24 hpf biological replicate 2 technical replicate 1 to 6; Danio rerio; OTHER", "GSM8327217 r1", "GSM8327217", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "pert_exp2_brep2_trep1_to_6_S0_L001_R1_001.fastq.gz pert_exp2_brep2_trep1_to_6_S0_L001_R2_001.fastq.gz pert_exp2_brep2_trep1_to_6_S0_L001_R3_001.fastq.gz pert_exp2_brep2_trep1_to_6_S0_L001_R4_001.fastq.gz", "fastq fastq fastq fastq", 9307639614.0, 102281754.0, "GSM8327217 r1", "0:61 1:8 2:8 3:14", "A:1721795009;C:1288169328;G:1244888947;T:1984236555;N:97155", 61, 8, 8, 14, 1721795009, 1288169328, 1244888947, 1984236555, 97155, "SRX24912668", "SRS21618601", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32738, "SRR29398877", "SRX24912668", "SRS21618601", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Perturbation experiment 2 24 hpf biological replicate 2 technical replicate 1 to 6", "GSM8327217", null, "source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf|geo loc name:missing|collection date:missing", "Perturbation experiment 2 24 hpf biological replicate 2 technical replicate 1 to 6", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf", "GSM8327217", "GSM8327217: Perturbation experiment 2 24 hpf biological replicate 2 technical replicate 1 to 6; Danio rerio; OTHER", "GSM8327217 r1", "GSM8327217", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "pert_exp2_brep2_trep1_to_6_S0_L002_R1_001.fastq.gz pert_exp2_brep2_trep1_to_6_S0_L002_R2_001.fastq.gz pert_exp2_brep2_trep1_to_6_S0_L002_R3_001.fastq.gz pert_exp2_brep2_trep1_to_6_S0_L002_R4_001.fastq.gz", "fastq fastq fastq fastq", 9113682123.0, 100150353.0, "GSM8327217 r2", "0:61 1:8 2:8 3:14", "A:1686097187;C:1259089439;G:1218258982;T:1945625439;N:100486", 61, 8, 8, 14, 1686097187, 1259089439, 1218258982, 1945625439, 100486, "SRX24912668", "SRS21618601", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32739, "SRR29398878", "SRX24912668", "SRS21618601", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Perturbation experiment 2 24 hpf biological replicate 2 technical replicate 1 to 6", "GSM8327217", null, "source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf|geo loc name:missing|collection date:missing", "Perturbation experiment 2 24 hpf biological replicate 2 technical replicate 1 to 6", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf", "GSM8327217", "GSM8327217: Perturbation experiment 2 24 hpf biological replicate 2 technical replicate 1 to 6; Danio rerio; OTHER", "GSM8327217 r1", "GSM8327217", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "pert_exp2_brep2_trep1_to_6_S0_L003_R1_001.fastq.gz pert_exp2_brep2_trep1_to_6_S0_L003_R2_001.fastq.gz pert_exp2_brep2_trep1_to_6_S0_L003_R3_001.fastq.gz pert_exp2_brep2_trep1_to_6_S0_L003_R4_001.fastq.gz", "fastq fastq fastq fastq", 9286087720.0, 102044920.0, "GSM8327217 r3", "0:61 1:8 2:8 3:14", "A:1716420906;C:1285666517;G:1243365905;T:1979187027;N:99765", 61, 8, 8, 14, 1716420906, 1285666517, 1243365905, 1979187027, 99765, "SRX24912668", "SRS21618601", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32740, "SRR29398879", "SRX24912668", "SRS21618601", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Perturbation experiment 2 24 hpf biological replicate 2 technical replicate 1 to 6", "GSM8327217", null, "source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf|geo loc name:missing|collection date:missing", "Perturbation experiment 2 24 hpf biological replicate 2 technical replicate 1 to 6", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf", "GSM8327217", "GSM8327217: Perturbation experiment 2 24 hpf biological replicate 2 technical replicate 1 to 6; Danio rerio; OTHER", "GSM8327217 r1", "GSM8327217", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "pert_exp2_brep2_trep1_to_6_S0_L004_R1_001.fastq.gz pert_exp2_brep2_trep1_to_6_S0_L004_R2_001.fastq.gz pert_exp2_brep2_trep1_to_6_S0_L004_R3_001.fastq.gz pert_exp2_brep2_trep1_to_6_S0_L004_R4_001.fastq.gz", "fastq fastq fastq fastq", 9274368740.0, 101916140.0, "GSM8327217 r4", "0:61 1:8 2:8 3:14", "A:1714501829;C:1283534271;G:1240916416;T:1977833740;N:98284", 61, 8, 8, 14, 1714501829, 1283534271, 1240916416, 1977833740, 98284, "SRX24912668", "SRS21618601", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32741, "SRR29398880", "SRX24912667", "SRS21618602", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Perturbation experiment 2 24 hpf biological replicate 1 technical replicate 1 to 4", "GSM8327216", null, "source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf|geo loc name:missing|collection date:missing", "Perturbation experiment 2 24 hpf biological replicate 1 technical replicate 1 to 4", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf", "GSM8327216", "GSM8327216: Perturbation experiment 2 24 hpf biological replicate 1 technical replicate 1 to 4; Danio rerio; OTHER", "GSM8327216 r1", "GSM8327216", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "pert_exp2_brep1_trep1_2_3_4_S0_L001_R1_001.fastq.gz pert_exp2_brep1_trep1_2_3_4_S0_L001_R2_001.fastq.gz pert_exp2_brep1_trep1_2_3_4_S0_L001_R3_001.fastq.gz pert_exp2_brep1_trep1_2_3_4_S0_L001_R4_001.fastq.gz", "fastq fastq fastq fastq", 10478915720.0, 115152920.0, "GSM8327216 r1", "0:61 1:8 2:8 3:14", "A:1736895270;C:1397254088;G:1357881943;T:2531770836;N:525983", 61, 8, 8, 14, 1736895270, 1397254088, 1357881943, 2531770836, 525983, "SRX24912667", "SRS21618602", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32742, "SRR29398881", "SRX24912667", "SRS21618602", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Perturbation experiment 2 24 hpf biological replicate 1 technical replicate 1 to 4", "GSM8327216", null, "source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf|geo loc name:missing|collection date:missing", "Perturbation experiment 2 24 hpf biological replicate 1 technical replicate 1 to 4", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf", "GSM8327216", "GSM8327216: Perturbation experiment 2 24 hpf biological replicate 1 technical replicate 1 to 4; Danio rerio; OTHER", "GSM8327216 r1", "GSM8327216", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "pert_exp2_brep1_trep1_2_3_4_S0_L002_R1_001.fastq.gz pert_exp2_brep1_trep1_2_3_4_S0_L002_R2_001.fastq.gz pert_exp2_brep1_trep1_2_3_4_S0_L002_R3_001.fastq.gz pert_exp2_brep1_trep1_2_3_4_S0_L002_R4_001.fastq.gz", "fastq fastq fastq fastq", 10976773171.0, 120623881.0, "GSM8327216 r2", "0:61 1:8 2:8 3:14", "A:1854295106;C:1459958363;G:1422957853;T:2620345368;N:500051", 61, 8, 8, 14, 1854295106, 1459958363, 1422957853, 2620345368, 500051, "SRX24912667", "SRS21618602", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32743, "SRR29398882", "SRX24912667", "SRS21618602", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Perturbation experiment 2 24 hpf biological replicate 1 technical replicate 1 to 4", "GSM8327216", null, "source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf|geo loc name:missing|collection date:missing", "Perturbation experiment 2 24 hpf biological replicate 1 technical replicate 1 to 4", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf", "GSM8327216", "GSM8327216: Perturbation experiment 2 24 hpf biological replicate 1 technical replicate 1 to 4; Danio rerio; OTHER", "GSM8327216 r1", "GSM8327216", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "pert_exp2_brep1_trep1_2_3_4_S0_L003_R1_001.fastq.gz pert_exp2_brep1_trep1_2_3_4_S0_L003_R2_001.fastq.gz pert_exp2_brep1_trep1_2_3_4_S0_L003_R3_001.fastq.gz pert_exp2_brep1_trep1_2_3_4_S0_L003_R4_001.fastq.gz", "fastq fastq fastq fastq", 10970493898.0, 120554878.0, "GSM8327216 r3", "0:61 1:8 2:8 3:14", "A:1804346523;C:1462166175;G:1421809080;T:2665109964;N:415816", 61, 8, 8, 14, 1804346523, 1462166175, 1421809080, 2665109964, 415816, "SRX24912667", "SRS21618602", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32744, "SRR29398883", "SRX24912667", "SRS21618602", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Perturbation experiment 2 24 hpf biological replicate 1 technical replicate 1 to 4", "GSM8327216", null, "source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf|geo loc name:missing|collection date:missing", "Perturbation experiment 2 24 hpf biological replicate 1 technical replicate 1 to 4", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf", "GSM8327216", "GSM8327216: Perturbation experiment 2 24 hpf biological replicate 1 technical replicate 1 to 4; Danio rerio; OTHER", "GSM8327216 r1", "GSM8327216", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "pert_exp2_brep1_trep1_2_3_4_S0_L004_R1_001.fastq.gz pert_exp2_brep1_trep1_2_3_4_S0_L004_R2_001.fastq.gz pert_exp2_brep1_trep1_2_3_4_S0_L004_R3_001.fastq.gz pert_exp2_brep1_trep1_2_3_4_S0_L004_R4_001.fastq.gz", "fastq fastq fastq fastq", 11081541198.0, 121775178.0, "GSM8327216 r4", "0:61 1:8 2:8 3:14", "A:1869228214;C:1474483692;G:1435361382;T:2648837266;N:375304", 61, 8, 8, 14, 1869228214, 1474483692, 1435361382, 2648837266, 375304, "SRX24912667", "SRS21618602", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32745, "SRR29398884", "SRX24912666", "SRS21618600", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Perturbation experiment 1 6 hpf 24 hpf biological replicate 3 technical replicate 1", "GSM8327215", null, "source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf|geo loc name:missing|collection date:missing", "Perturbation experiment 1 6 hpf 24 hpf biological replicate 3 technical replicate 1", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf", "GSM8327215", "GSM8327215: Perturbation experiment 1 6 hpf 24 hpf biological replicate 3 technical replicate 1; Danio rerio; OTHER", "GSM8327215 r1", "GSM8327215", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "pert_exp1_brep3_trep1_S0_L001_R1_001.fastq.gz pert_exp1_brep3_trep1_S0_L001_R2_001.fastq.gz pert_exp1_brep3_trep1_S0_L001_R3_001.fastq.gz pert_exp1_brep3_trep1_S0_L001_R4_001.fastq.gz", "fastq fastq fastq fastq", 9825258079.0, 107969869.0, "GSM8327215 r1", "0:61 1:8 2:8 3:14", "A:1892998816;C:1448298491;G:1287308183;T:1956393917;N:1162602", 61, 8, 8, 14, 1892998816, 1448298491, 1287308183, 1956393917, 1162602, "SRX24912666", "SRS21618600", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32746, "SRR29398885", "SRX24912666", "SRS21618600", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Perturbation experiment 1 6 hpf 24 hpf biological replicate 3 technical replicate 1", "GSM8327215", null, "source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf|geo loc name:missing|collection date:missing", "Perturbation experiment 1 6 hpf 24 hpf biological replicate 3 technical replicate 1", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf", "GSM8327215", "GSM8327215: Perturbation experiment 1 6 hpf 24 hpf biological replicate 3 technical replicate 1; Danio rerio; OTHER", "GSM8327215 r1", "GSM8327215", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "pert_exp1_brep3_trep1_S0_L002_R1_001.fastq.gz pert_exp1_brep3_trep1_S0_L002_R2_001.fastq.gz pert_exp1_brep3_trep1_S0_L002_R3_001.fastq.gz pert_exp1_brep3_trep1_S0_L002_R4_001.fastq.gz", "fastq fastq fastq fastq", 9797467225.0, 107664475.0, "GSM8327215 r2", "0:61 1:8 2:8 3:14", "A:1884493825;C:1436463441;G:1294831461;T:1950665696;N:1078552", 61, 8, 8, 14, 1884493825, 1436463441, 1294831461, 1950665696, 1078552, "SRX24912666", "SRS21618600", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32747, "SRR29398886", "SRX24912666", "SRS21618600", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Perturbation experiment 1 6 hpf 24 hpf biological replicate 3 technical replicate 1", "GSM8327215", null, "source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf|geo loc name:missing|collection date:missing", "Perturbation experiment 1 6 hpf 24 hpf biological replicate 3 technical replicate 1", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf", "GSM8327215", "GSM8327215: Perturbation experiment 1 6 hpf 24 hpf biological replicate 3 technical replicate 1; Danio rerio; OTHER", "GSM8327215 r1", "GSM8327215", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "pert_exp1_brep3_trep1_S0_L003_R1_001.fastq.gz pert_exp1_brep3_trep1_S0_L003_R2_001.fastq.gz pert_exp1_brep3_trep1_S0_L003_R3_001.fastq.gz pert_exp1_brep3_trep1_S0_L003_R4_001.fastq.gz", "fastq fastq fastq fastq", 9945377715.0, 109289865.0, "GSM8327215 r3", "0:61 1:8 2:8 3:14", "A:1919403458;C:1456796407;G:1307295125;T:1982126987;N:1059788", 61, 8, 8, 14, 1919403458, 1456796407, 1307295125, 1982126987, 1059788, "SRX24912666", "SRS21618600", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32748, "SRR29398887", "SRX24912666", "SRS21618600", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Perturbation experiment 1 6 hpf 24 hpf biological replicate 3 technical replicate 1", "GSM8327215", null, "source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf|geo loc name:missing|collection date:missing", "Perturbation experiment 1 6 hpf 24 hpf biological replicate 3 technical replicate 1", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf", "GSM8327215", "GSM8327215: Perturbation experiment 1 6 hpf 24 hpf biological replicate 3 technical replicate 1; Danio rerio; OTHER", "GSM8327215 r1", "GSM8327215", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "pert_exp1_brep3_trep1_S0_L004_R1_001.fastq.gz pert_exp1_brep3_trep1_S0_L004_R2_001.fastq.gz pert_exp1_brep3_trep1_S0_L004_R3_001.fastq.gz pert_exp1_brep3_trep1_S0_L004_R4_001.fastq.gz", "fastq fastq fastq fastq", 9799158005.0, 107683055.0, "GSM8327215 r4", "0:61 1:8 2:8 3:14", "A:1887332404;C:1443564157;G:1288767599;T:1948048686;N:953509", 61, 8, 8, 14, 1887332404, 1443564157, 1288767599, 1948048686, 953509, "SRX24912666", "SRS21618600", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32749, "SRR29398888", "SRX24912665", "SRS21618599", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Perturbation experiment 1 6 hpf 14 hpf biological replicate 1 technical replicate 1", "GSM8327213", null, "source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf|geo loc name:missing|collection date:missing", "Perturbation experiment 1 6 hpf 14 hpf biological replicate 1 technical replicate 1", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf", "GSM8327213", "GSM8327213: Perturbation experiment 1 6 hpf 14 hpf biological replicate 1 technical replicate 1; Danio rerio; OTHER", "GSM8327213 r1", "GSM8327213", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "pert_exp1_brep1_trep1_S0_L001_R1_001_run1.fastq.gz pert_exp1_brep1_trep1_S0_L001_R2_001_run1.fastq.gz pert_exp1_brep1_trep1_S0_L001_R3_001_run1.fastq.gz pert_exp1_brep1_trep1_S0_L001_R4_001_run1.fastq.gz", "fastq fastq fastq fastq", 5753105540.0, 63220940.0, "GSM8327213 r1", "0:61 1:8 2:8 3:14", "A:1174237214;C:759555395;G:705449866;T:1217212379;N:22486", 61, 8, 8, 14, 1174237214, 759555395, 705449866, 1217212379, 22486, "SRX24912665", "SRS21618599", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32750, "SRR29398889", "SRX24912665", "SRS21618599", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Perturbation experiment 1 6 hpf 14 hpf biological replicate 1 technical replicate 1", "GSM8327213", null, "source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf|geo loc name:missing|collection date:missing", "Perturbation experiment 1 6 hpf 14 hpf biological replicate 1 technical replicate 1", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf", "GSM8327213", "GSM8327213: Perturbation experiment 1 6 hpf 14 hpf biological replicate 1 technical replicate 1; Danio rerio; OTHER", "GSM8327213 r1", "GSM8327213", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "pert_exp1_brep1_trep1_S0_L002_R1_001_run1.fastq.gz pert_exp1_brep1_trep1_S0_L002_R2_001_run1.fastq.gz pert_exp1_brep1_trep1_S0_L002_R3_001_run1.fastq.gz pert_exp1_brep1_trep1_S0_L002_R4_001_run1.fastq.gz", "fastq fastq fastq fastq", 5588689379.0, 61414169.0, "GSM8327213 r2", "0:61 1:8 2:8 3:14", "A:1135353331;C:739291995;G:689386853;T:1182212845;N:19285", 61, 8, 8, 14, 1135353331, 739291995, 689386853, 1182212845, 19285, "SRX24912665", "SRS21618599", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32751, "SRR29398890", "SRX24912665", "SRS21618599", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Perturbation experiment 1 6 hpf 14 hpf biological replicate 1 technical replicate 1", "GSM8327213", null, "source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf|geo loc name:missing|collection date:missing", "Perturbation experiment 1 6 hpf 14 hpf biological replicate 1 technical replicate 1", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf", "GSM8327213", "GSM8327213: Perturbation experiment 1 6 hpf 14 hpf biological replicate 1 technical replicate 1; Danio rerio; OTHER", "GSM8327213 r1", "GSM8327213", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "pert_exp1_brep1_trep1_S0_L003_R1_001_run1.fastq.gz pert_exp1_brep1_trep1_S0_L003_R2_001_run1.fastq.gz pert_exp1_brep1_trep1_S0_L003_R3_001_run1.fastq.gz pert_exp1_brep1_trep1_S0_L003_R4_001_run1.fastq.gz", "fastq fastq fastq fastq", 5731949769.0, 62988459.0, "GSM8327213 r3", "0:61 1:8 2:8 3:14", "A:1167937533;C:754846871;G:708985526;T:1210491951;N:34118", 61, 8, 8, 14, 1167937533, 754846871, 708985526, 1210491951, 34118, "SRX24912665", "SRS21618599", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32752, "SRR29398891", "SRX24912665", "SRS21618599", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Perturbation experiment 1 6 hpf 14 hpf biological replicate 1 technical replicate 1", "GSM8327213", null, "source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf|geo loc name:missing|collection date:missing", "Perturbation experiment 1 6 hpf 14 hpf biological replicate 1 technical replicate 1", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf", "GSM8327213", "GSM8327213: Perturbation experiment 1 6 hpf 14 hpf biological replicate 1 technical replicate 1; Danio rerio; OTHER", "GSM8327213 r1", "GSM8327213", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "pert_exp1_brep1_trep1_S0_L002_R1_001_run2.fastq.gz pert_exp1_brep1_trep1_S0_L002_R2_001_run2.fastq.gz pert_exp1_brep1_trep1_S0_L002_R3_001_run2.fastq.gz pert_exp1_brep1_trep1_S0_L002_R4_001_run2.fastq.gz", "fastq fastq fastq fastq", 7183714902.0, 78941922.0, "GSM8327213 r6", "0:61 1:8 2:8 3:14", "A:1413777790;C:968745759;G:906288455;T:1526622859;N:22379", 61, 8, 8, 14, 1413777790, 968745759, 906288455, 1526622859, 22379, "SRX24912665", "SRS21618599", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32753, "SRR29398892", "SRX24912665", "SRS21618599", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Perturbation experiment 1 6 hpf 14 hpf biological replicate 1 technical replicate 1", "GSM8327213", null, "source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf|geo loc name:missing|collection date:missing", "Perturbation experiment 1 6 hpf 14 hpf biological replicate 1 technical replicate 1", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf", "GSM8327213", "GSM8327213: Perturbation experiment 1 6 hpf 14 hpf biological replicate 1 technical replicate 1; Danio rerio; OTHER", "GSM8327213 r1", "GSM8327213", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "pert_exp1_brep1_trep1_S0_L003_R1_001_run2.fastq.gz pert_exp1_brep1_trep1_S0_L003_R2_001_run2.fastq.gz pert_exp1_brep1_trep1_S0_L003_R3_001_run2.fastq.gz pert_exp1_brep1_trep1_S0_L003_R4_001_run2.fastq.gz", "fastq fastq fastq fastq", 7347837315.0, 80745465.0, "GSM8327213 r7", "0:61 1:8 2:8 3:14", "A:1442258471;C:999458019;G:933530375;T:1550199068;N:27432", 61, 8, 8, 14, 1442258471, 999458019, 933530375, 1550199068, 27432, "SRX24912665", "SRS21618599", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32754, "SRR29398893", "SRX24912665", "SRS21618599", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Perturbation experiment 1 6 hpf 14 hpf biological replicate 1 technical replicate 1", "GSM8327213", null, "source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf|geo loc name:missing|collection date:missing", "Perturbation experiment 1 6 hpf 14 hpf biological replicate 1 technical replicate 1", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf", "GSM8327213", "GSM8327213: Perturbation experiment 1 6 hpf 14 hpf biological replicate 1 technical replicate 1; Danio rerio; OTHER", "GSM8327213 r1", "GSM8327213", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "pert_exp1_brep1_trep1_S0_L004_R1_001_run2.fastq.gz pert_exp1_brep1_trep1_S0_L004_R2_001_run2.fastq.gz pert_exp1_brep1_trep1_S0_L004_R3_001_run2.fastq.gz pert_exp1_brep1_trep1_S0_L004_R4_001_run2.fastq.gz", "fastq fastq fastq fastq", 7549030853.0, 82956383.0, "GSM8327213 r8", "0:61 1:8 2:8 3:14", "A:1483634179;C:1026783953;G:942155543;T:1607740711;N:24977", 61, 8, 8, 14, 1483634179, 1026783953, 942155543, 1607740711, 24977, "SRX24912665", "SRS21618599", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32755, "SRR29398906", "SRX24912665", "SRS21618599", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Perturbation experiment 1 6 hpf 14 hpf biological replicate 1 technical replicate 1", "GSM8327213", null, "source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf|geo loc name:missing|collection date:missing", "Perturbation experiment 1 6 hpf 14 hpf biological replicate 1 technical replicate 1", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf", "GSM8327213", "GSM8327213: Perturbation experiment 1 6 hpf 14 hpf biological replicate 1 technical replicate 1; Danio rerio; OTHER", "GSM8327213 r1", "GSM8327213", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "pert_exp1_brep1_trep1_S0_L004_R1_001_run1.fastq.gz pert_exp1_brep1_trep1_S0_L004_R2_001_run1.fastq.gz pert_exp1_brep1_trep1_S0_L004_R3_001_run1.fastq.gz pert_exp1_brep1_trep1_S0_L004_R4_001_run1.fastq.gz", "fastq fastq fastq fastq", 5617759875.0, 61733625.0, "GSM8327213 r4", "0:61 1:8 2:8 3:14", "A:1141997479;C:743305987;G:692463270;T:1187966465;N:17924", 61, 8, 8, 14, 1141997479, 743305987, 692463270, 1187966465, 17924, "SRX24912665", "SRS21618599", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32756, "SRR29398907", "SRX24912665", "SRS21618599", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Perturbation experiment 1 6 hpf 14 hpf biological replicate 1 technical replicate 1", "GSM8327213", null, "source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf|geo loc name:missing|collection date:missing", "Perturbation experiment 1 6 hpf 14 hpf biological replicate 1 technical replicate 1", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf", "GSM8327213", "GSM8327213: Perturbation experiment 1 6 hpf 14 hpf biological replicate 1 technical replicate 1; Danio rerio; OTHER", "GSM8327213 r1", "GSM8327213", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "pert_exp1_brep1_trep1_S0_L001_R1_001_run2.fastq.gz pert_exp1_brep1_trep1_S0_L001_R2_001_run2.fastq.gz pert_exp1_brep1_trep1_S0_L001_R3_001_run2.fastq.gz pert_exp1_brep1_trep1_S0_L001_R4_001_run2.fastq.gz", "fastq fastq fastq fastq", 7274157527.0, 79935797.0, "GSM8327213 r5", "0:61 1:8 2:8 3:14", "A:1431076165;C:991014006;G:919281150;T:1534687447;N:24849", 61, 8, 8, 14, 1431076165, 991014006, 919281150, 1534687447, 24849, "SRX24912665", "SRS21618599", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32757, "SRR29398894", "SRX24912664", "SRS21618598", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Perturbation experiment 1 6 hpf 24 hpf biological replicate 2 technical replicate 1", "GSM8327214", null, "source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf|geo loc name:missing|collection date:missing", "Perturbation experiment 1 6 hpf 24 hpf biological replicate 2 technical replicate 1", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf", "GSM8327214", "GSM8327214: Perturbation experiment 1 6 hpf 24 hpf biological replicate 2 technical replicate 1; Danio rerio; OTHER", "GSM8327214 r1", "GSM8327214", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "pert_exp1_brep2_trep1_S0_L001_R1_001_run1.fastq.gz pert_exp1_brep2_trep1_S0_L001_R2_001_run1.fastq.gz pert_exp1_brep2_trep1_S0_L001_R3_001_run1.fastq.gz pert_exp1_brep2_trep1_S0_L001_R4_001_run1.fastq.gz", "fastq fastq fastq fastq", 10423478247.0, 114543717.0, "GSM8327214 r1", "0:61 1:8 2:8 3:14", "A:2025794098;C:1533084111;G:1357456923;T:2070555537;N:276068", 61, 8, 8, 14, 2025794098, 1533084111, 1357456923, 2070555537, 276068, "SRX24912664", "SRS21618598", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32758, "SRR29398895", "SRX24912664", "SRS21618598", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Perturbation experiment 1 6 hpf 24 hpf biological replicate 2 technical replicate 1", "GSM8327214", null, "source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf|geo loc name:missing|collection date:missing", "Perturbation experiment 1 6 hpf 24 hpf biological replicate 2 technical replicate 1", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf", "GSM8327214", "GSM8327214: Perturbation experiment 1 6 hpf 24 hpf biological replicate 2 technical replicate 1; Danio rerio; OTHER", "GSM8327214 r1", "GSM8327214", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "pert_exp1_brep2_trep1_S0_L002_R1_001_run1.fastq.gz pert_exp1_brep2_trep1_S0_L002_R2_001_run1.fastq.gz pert_exp1_brep2_trep1_S0_L002_R3_001_run1.fastq.gz pert_exp1_brep2_trep1_S0_L002_R4_001_run1.fastq.gz", "fastq fastq fastq fastq", 10655252153.0, 117090683.0, "GSM8327214 r2", "0:61 1:8 2:8 3:14", "A:2076209515;C:1557182701;G:1384355121;T:2124543476;N:240850", 61, 8, 8, 14, 2076209515, 1557182701, 1384355121, 2124543476, 240850, "SRX24912664", "SRS21618598", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32759, "SRR29398896", "SRX24912664", "SRS21618598", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Perturbation experiment 1 6 hpf 24 hpf biological replicate 2 technical replicate 1", "GSM8327214", null, "source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf|geo loc name:missing|collection date:missing", "Perturbation experiment 1 6 hpf 24 hpf biological replicate 2 technical replicate 1", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf", "GSM8327214", "GSM8327214: Perturbation experiment 1 6 hpf 24 hpf biological replicate 2 technical replicate 1; Danio rerio; OTHER", "GSM8327214 r1", "GSM8327214", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "pert_exp1_brep2_trep1_S0_L003_R1_001_run1.fastq.gz pert_exp1_brep2_trep1_S0_L003_R2_001_run1.fastq.gz pert_exp1_brep2_trep1_S0_L003_R3_001_run1.fastq.gz pert_exp1_brep2_trep1_S0_L003_R4_001_run1.fastq.gz", "fastq fastq fastq fastq", 10684967475.0, 117417225.0, "GSM8327214 r3", "0:61 1:8 2:8 3:14", "A:2080492208;C:1561134821;G:1396957165;T:2123612525;N:254006", 61, 8, 8, 14, 2080492208, 1561134821, 1396957165, 2123612525, 254006, "SRX24912664", "SRS21618598", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32760, "SRR29398897", "SRX24912664", "SRS21618598", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Perturbation experiment 1 6 hpf 24 hpf biological replicate 2 technical replicate 1", "GSM8327214", null, "source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf|geo loc name:missing|collection date:missing", "Perturbation experiment 1 6 hpf 24 hpf biological replicate 2 technical replicate 1", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf", "GSM8327214", "GSM8327214: Perturbation experiment 1 6 hpf 24 hpf biological replicate 2 technical replicate 1; Danio rerio; OTHER", "GSM8327214 r1", "GSM8327214", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "pert_exp1_brep2_trep1_S0_L004_R1_001_run1.fastq.gz pert_exp1_brep2_trep1_S0_L004_R2_001_run1.fastq.gz pert_exp1_brep2_trep1_S0_L004_R3_001_run1.fastq.gz pert_exp1_brep2_trep1_S0_L004_R4_001_run1.fastq.gz", "fastq fastq fastq fastq", 10707189584.0, 117661424.0, "GSM8327214 r4", "0:61 1:8 2:8 3:14", "A:2083689453;C:1573371636;G:1391615902;T:2128351952;N:317921", 61, 8, 8, 14, 2083689453, 1573371636, 1391615902, 2128351952, 317921, "SRX24912664", "SRS21618598", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32761, "SRR29398898", "SRX24912664", "SRS21618598", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Perturbation experiment 1 6 hpf 24 hpf biological replicate 2 technical replicate 1", "GSM8327214", null, "source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf|geo loc name:missing|collection date:missing", "Perturbation experiment 1 6 hpf 24 hpf biological replicate 2 technical replicate 1", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf", "GSM8327214", "GSM8327214: Perturbation experiment 1 6 hpf 24 hpf biological replicate 2 technical replicate 1; Danio rerio; OTHER", "GSM8327214 r1", "GSM8327214", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "pert_exp1_brep2_trep1_S0_L001_R1_001_run2.fastq.gz pert_exp1_brep2_trep1_S0_L001_R2_001_run2.fastq.gz pert_exp1_brep2_trep1_S0_L001_R3_001_run2.fastq.gz pert_exp1_brep2_trep1_S0_L001_R4_001_run2.fastq.gz", "fastq fastq fastq fastq", 11030224205.0, 121211255.0, "GSM8327214 r5", "0:61 1:8 2:8 3:14", "A:2183422666;C:1601517274;G:1426241368;T:2178932541;N:3772706", 61, 8, 8, 14, 2183422666, 1601517274, 1426241368, 2178932541, 3772706, "SRX24912664", "SRS21618598", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32762, "SRR29398899", "SRX24912664", "SRS21618598", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Perturbation experiment 1 6 hpf 24 hpf biological replicate 2 technical replicate 1", "GSM8327214", null, "source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf|geo loc name:missing|collection date:missing", "Perturbation experiment 1 6 hpf 24 hpf biological replicate 2 technical replicate 1", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf", "GSM8327214", "GSM8327214: Perturbation experiment 1 6 hpf 24 hpf biological replicate 2 technical replicate 1; Danio rerio; OTHER", "GSM8327214 r1", "GSM8327214", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "pert_exp1_brep2_trep1_S0_L002_R1_001_run2.fastq.gz pert_exp1_brep2_trep1_S0_L002_R2_001_run2.fastq.gz pert_exp1_brep2_trep1_S0_L002_R3_001_run2.fastq.gz pert_exp1_brep2_trep1_S0_L002_R4_001_run2.fastq.gz", "fastq fastq fastq fastq", 11030253234.0, 121211574.0, "GSM8327214 r6", "0:61 1:8 2:8 3:14", "A:2188443587;C:1589039147;G:1426719226;T:2185985975;N:3718079", 61, 8, 8, 14, 2188443587, 1589039147, 1426719226, 2185985975, 3718079, "SRX24912664", "SRS21618598", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32763, "SRR29398900", "SRX24912664", "SRS21618598", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Perturbation experiment 1 6 hpf 24 hpf biological replicate 2 technical replicate 1", "GSM8327214", null, "source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf|geo loc name:missing|collection date:missing", "Perturbation experiment 1 6 hpf 24 hpf biological replicate 2 technical replicate 1", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf", "GSM8327214", "GSM8327214: Perturbation experiment 1 6 hpf 24 hpf biological replicate 2 technical replicate 1; Danio rerio; OTHER", "GSM8327214 r1", "GSM8327214", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "pert_exp1_brep2_trep1_S0_L003_R1_001_run2.fastq.gz pert_exp1_brep2_trep1_S0_L003_R2_001_run2.fastq.gz pert_exp1_brep2_trep1_S0_L003_R3_001_run2.fastq.gz pert_exp1_brep2_trep1_S0_L003_R4_001_run2.fastq.gz", "fastq fastq fastq fastq", 11084660405.0, 121809455.0, "GSM8327214 r7", "0:61 1:8 2:8 3:14", "A:2203007980;C:1596382135;G:1428403216;T:2199749125;N:2834299", 61, 8, 8, 14, 2203007980, 1596382135, 1428403216, 2199749125, 2834299, "SRX24912664", "SRS21618598", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32764, "SRR29398901", "SRX24912664", "SRS21618598", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Perturbation experiment 1 6 hpf 24 hpf biological replicate 2 technical replicate 1", "GSM8327214", null, "source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf|geo loc name:missing|collection date:missing", "Perturbation experiment 1 6 hpf 24 hpf biological replicate 2 technical replicate 1", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 6 hpf", "GSM8327214", "GSM8327214: Perturbation experiment 1 6 hpf 24 hpf biological replicate 2 technical replicate 1; Danio rerio; OTHER", "GSM8327214 r1", "GSM8327214", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "pert_exp1_brep2_trep1_S0_L004_R1_001_run2.fastq.gz pert_exp1_brep2_trep1_S0_L004_R2_001_run2.fastq.gz pert_exp1_brep2_trep1_S0_L004_R3_001_run2.fastq.gz pert_exp1_brep2_trep1_S0_L004_R4_001_run2.fastq.gz", "fastq fastq fastq fastq", 11063671801.0, 121578811.0, "GSM8327214 r8", "0:61 1:8 2:8 3:14", "A:2194629946;C:1601965741;G:1426892122;T:2190032980;N:2786682", 61, 8, 8, 14, 2194629946, 1601965741, 1426892122, 2190032980, 2786682, "SRX24912664", "SRS21618598", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32765, "SRR29398902", "SRX24912663", "SRS21618597", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Reference experiment 14 hpf 24 hpf biological replicate 2 and 3 technical replicate 2", "GSM8327212", null, "source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 14 hpf|geo loc name:missing|collection date:missing", "Reference experiment 14 hpf 24 hpf biological replicate 2 and 3 technical replicate 2", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 14 hpf", "GSM8327212", "GSM8327212: Reference experiment 14 hpf 24 hpf biological replicate 2 and 3 technical replicate 2; Danio rerio; OTHER", "GSM8327212 r1", "GSM8327212", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "ref_exp_brep2_3_trep2_S0_L001_R1_001.fastq.gz ref_exp_brep2_3_trep2_S0_L001_R2_001.fastq.gz ref_exp_brep2_3_trep2_S0_L001_R3_001.fastq.gz ref_exp_brep2_3_trep2_S0_L001_R4_001.fastq.gz", "fastq fastq fastq fastq", 12384624343.0, 136094773.0, "GSM8327212 r1", "0:61 1:8 2:8 3:14", "A:2294297792;C:1657100436;G:1635865148;T:2714342859;N:174918", 61, 8, 8, 14, 2294297792, 1657100436, 1635865148, 2714342859, 174918, "SRX24912663", "SRS21618597", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32766, "SRR29398903", "SRX24912663", "SRS21618597", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Reference experiment 14 hpf 24 hpf biological replicate 2 and 3 technical replicate 2", "GSM8327212", null, "source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 14 hpf|geo loc name:missing|collection date:missing", "Reference experiment 14 hpf 24 hpf biological replicate 2 and 3 technical replicate 2", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 14 hpf", "GSM8327212", "GSM8327212: Reference experiment 14 hpf 24 hpf biological replicate 2 and 3 technical replicate 2; Danio rerio; OTHER", "GSM8327212 r1", "GSM8327212", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "ref_exp_brep2_3_trep2_S0_L002_R1_001.fastq.gz ref_exp_brep2_3_trep2_S0_L002_R2_001.fastq.gz ref_exp_brep2_3_trep2_S0_L002_R3_001.fastq.gz ref_exp_brep2_3_trep2_S0_L002_R4_001.fastq.gz", "fastq fastq fastq fastq", 12315448600.0, 135334600.0, "GSM8327212 r2", "0:61 1:8 2:8 3:14", "A:2283746259;C:1642777354;G:1624372665;T:2704362532;N:151790", 61, 8, 8, 14, 2283746259, 1642777354, 1624372665, 2704362532, 151790, "SRX24912663", "SRS21618597", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32767, "SRR29398904", "SRX24912663", "SRS21618597", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Reference experiment 14 hpf 24 hpf biological replicate 2 and 3 technical replicate 2", "GSM8327212", null, "source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 14 hpf|geo loc name:missing|collection date:missing", "Reference experiment 14 hpf 24 hpf biological replicate 2 and 3 technical replicate 2", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 14 hpf", "GSM8327212", "GSM8327212: Reference experiment 14 hpf 24 hpf biological replicate 2 and 3 technical replicate 2; Danio rerio; OTHER", "GSM8327212 r1", "GSM8327212", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "ref_exp_brep2_3_trep2_S0_L003_R1_001.fastq.gz ref_exp_brep2_3_trep2_S0_L003_R2_001.fastq.gz ref_exp_brep2_3_trep2_S0_L003_R3_001.fastq.gz ref_exp_brep2_3_trep2_S0_L003_R4_001.fastq.gz", "fastq fastq fastq fastq", 12523818580.0, 137624380.0, "GSM8327212 r3", "0:61 1:8 2:8 3:14", "A:2324226717;C:1670367024;G:1657065051;T:2743216042;N:212346", 61, 8, 8, 14, 2324226717, 1670367024, 1657065051, 2743216042, 212346, "SRX24912663", "SRS21618597", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32768, "SRR29398905", "SRX24912663", "SRS21618597", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Reference experiment 14 hpf 24 hpf biological replicate 2 and 3 technical replicate 2", "GSM8327212", null, "source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 14 hpf|geo loc name:missing|collection date:missing", "Reference experiment 14 hpf 24 hpf biological replicate 2 and 3 technical replicate 2", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 14 hpf", "GSM8327212", "GSM8327212: Reference experiment 14 hpf 24 hpf biological replicate 2 and 3 technical replicate 2; Danio rerio; OTHER", "GSM8327212 r1", "GSM8327212", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "ref_exp_brep2_3_trep2_S0_L004_R1_001.fastq.gz ref_exp_brep2_3_trep2_S0_L004_R2_001.fastq.gz ref_exp_brep2_3_trep2_S0_L004_R3_001.fastq.gz ref_exp_brep2_3_trep2_S0_L004_R4_001.fastq.gz", "fastq fastq fastq fastq", 12439046074.0, 136692814.0, "GSM8327212 r4", "0:61 1:8 2:8 3:14", "A:2308308565;C:1661073675;G:1642305225;T:2726252574;N:321615", 61, 8, 8, 14, 2308308565, 1661073675, 1642305225, 2726252574, 321615, "SRX24912663", "SRS21618597", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32769, "SRR29398908", "SRX24912662", "SRS21618595", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Reference experiment 14 hpf 24 hpf biological replicate 1 technical replicate 2", "GSM8327211", null, "source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 14 hpf|geo loc name:missing|collection date:missing", "Reference experiment 14 hpf 24 hpf biological replicate 1 technical replicate 2", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 14 hpf", "GSM8327211", "GSM8327211: Reference experiment 14 hpf 24 hpf biological replicate 1 technical replicate 2; Danio rerio; OTHER", "GSM8327211 r1", "GSM8327211", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "ref_exp_brep1_trep2_S0_L001_R1_001.fastq.gz ref_exp_brep1_trep2_S0_L001_R2_001.fastq.gz ref_exp_brep1_trep2_S0_L001_R3_001.fastq.gz ref_exp_brep1_trep2_S0_L001_R4_001.fastq.gz", "fastq fastq fastq fastq", 13075759970.0, 143689670.0, "GSM8327211 r1", "0:61 1:8 2:8 3:14", "A:2455617490;C:1722060255;G:1704083809;T:2882118632;N:1189684", 61, 8, 8, 14, 2455617490, 1722060255, 1704083809, 2882118632, 1189684, "SRX24912662", "SRS21618595", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32770, "SRR29398909", "SRX24912662", "SRS21618595", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Reference experiment 14 hpf 24 hpf biological replicate 1 technical replicate 2", "GSM8327211", null, "source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 14 hpf|geo loc name:missing|collection date:missing", "Reference experiment 14 hpf 24 hpf biological replicate 1 technical replicate 2", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 14 hpf", "GSM8327211", "GSM8327211: Reference experiment 14 hpf 24 hpf biological replicate 1 technical replicate 2; Danio rerio; OTHER", "GSM8327211 r1", "GSM8327211", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "ref_exp_brep1_trep2_S0_L002_R1_001.fastq.gz ref_exp_brep1_trep2_S0_L002_R2_001.fastq.gz ref_exp_brep1_trep2_S0_L002_R3_001.fastq.gz ref_exp_brep1_trep2_S0_L002_R4_001.fastq.gz", "fastq fastq fastq fastq", 12965375878.0, 142476658.0, "GSM8327211 r2", "0:61 1:8 2:8 3:14", "A:2436528726;C:1704297673;G:1691673766;T:2857349303;N:1226670", 61, 8, 8, 14, 2436528726, 1704297673, 1691673766, 2857349303, 1226670, "SRX24912662", "SRS21618595", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32771, "SRR29398910", "SRX24912662", "SRS21618595", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Reference experiment 14 hpf 24 hpf biological replicate 1 technical replicate 2", "GSM8327211", null, "source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 14 hpf|geo loc name:missing|collection date:missing", "Reference experiment 14 hpf 24 hpf biological replicate 1 technical replicate 2", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 14 hpf", "GSM8327211", "GSM8327211: Reference experiment 14 hpf 24 hpf biological replicate 1 technical replicate 2; Danio rerio; OTHER", "GSM8327211 r1", "GSM8327211", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "ref_exp_brep1_trep2_S0_L003_R1_001.fastq.gz ref_exp_brep1_trep2_S0_L003_R2_001.fastq.gz ref_exp_brep1_trep2_S0_L003_R3_001.fastq.gz ref_exp_brep1_trep2_S0_L003_R4_001.fastq.gz", "fastq fastq fastq fastq", 13020225309.0, 143079399.0, "GSM8327211 r3", "0:61 1:8 2:8 3:14", "A:2448925813;C:1710138232;G:1703095600;T:2864268220;N:1415474", 61, 8, 8, 14, 2448925813, 1710138232, 1703095600, 2864268220, 1415474, "SRX24912662", "SRS21618595", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32772, "SRR29398911", "SRX24912662", "SRS21618595", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Reference experiment 14 hpf 24 hpf biological replicate 1 technical replicate 2", "GSM8327211", null, "source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 14 hpf|geo loc name:missing|collection date:missing", "Reference experiment 14 hpf 24 hpf biological replicate 1 technical replicate 2", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 14 hpf", "GSM8327211", "GSM8327211: Reference experiment 14 hpf 24 hpf biological replicate 1 technical replicate 2; Danio rerio; OTHER", "GSM8327211 r1", "GSM8327211", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "ref_exp_brep1_trep2_S0_L004_R1_001.fastq.gz ref_exp_brep1_trep2_S0_L004_R2_001.fastq.gz ref_exp_brep1_trep2_S0_L004_R3_001.fastq.gz ref_exp_brep1_trep2_S0_L004_R4_001.fastq.gz", "fastq fastq fastq fastq", 12968634588.0, 142512468.0, "GSM8327211 r4", "0:61 1:8 2:8 3:14", "A:2438050928;C:1706654424;G:1693567829;T:2853585948;N:1401419", 61, 8, 8, 14, 2438050928, 1706654424, 1693567829, 2853585948, 1401419, "SRX24912662", "SRS21618595", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32773, "SRR29398912", "SRX24912661", "SRS21618596", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Reference experiment 14 hpf 24 hpf biological replicate 2 and 3 technical replicate 1", "GSM8327210", null, "source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 14 hpf|geo loc name:missing|collection date:missing", "Reference experiment 14 hpf 24 hpf biological replicate 2 and 3 technical replicate 1", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 14 hpf", "GSM8327210", "GSM8327210: Reference experiment 14 hpf 24 hpf biological replicate 2 and 3 technical replicate 1; Danio rerio; OTHER", "GSM8327210 r1", "GSM8327210", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "ref_exp_brep2_3_trep1_S0_L001_R1_001.fastq.gz ref_exp_brep2_3_trep1_S0_L001_R2_001.fastq.gz ref_exp_brep2_3_trep1_S0_L001_R3_001.fastq.gz ref_exp_brep2_3_trep1_S0_L001_R4_001.fastq.gz", "fastq fastq fastq fastq", 10388141764.0, 114155404.0, "GSM8327210 r1", "0:61 1:8 2:8 3:14", "A:1922552965;C:1416885557;G:1386861514;T:2236850768;N:328840", 61, 8, 8, 14, 1922552965, 1416885557, 1386861514, 2236850768, 328840, "SRX24912661", "SRS21618596", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32774, "SRR29398913", "SRX24912661", "SRS21618596", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Reference experiment 14 hpf 24 hpf biological replicate 2 and 3 technical replicate 1", "GSM8327210", null, "source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 14 hpf|geo loc name:missing|collection date:missing", "Reference experiment 14 hpf 24 hpf biological replicate 2 and 3 technical replicate 1", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 14 hpf", "GSM8327210", "GSM8327210: Reference experiment 14 hpf 24 hpf biological replicate 2 and 3 technical replicate 1; Danio rerio; OTHER", "GSM8327210 r1", "GSM8327210", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "ref_exp_brep2_3_trep1_S0_L002_R1_001.fastq.gz ref_exp_brep2_3_trep1_S0_L002_R2_001.fastq.gz ref_exp_brep2_3_trep1_S0_L002_R3_001.fastq.gz ref_exp_brep2_3_trep1_S0_L002_R4_001.fastq.gz", "fastq fastq fastq fastq", 10440407978.0, 114729758.0, "GSM8327210 r2", "0:61 1:8 2:8 3:14", "A:1932601162;C:1422438299;G:1388104862;T:2254966360;N:404555", 61, 8, 8, 14, 1932601162, 1422438299, 1388104862, 2254966360, 404555, "SRX24912661", "SRS21618596", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32775, "SRR29398914", "SRX24912661", "SRS21618596", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Reference experiment 14 hpf 24 hpf biological replicate 2 and 3 technical replicate 1", "GSM8327210", null, "source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 14 hpf|geo loc name:missing|collection date:missing", "Reference experiment 14 hpf 24 hpf biological replicate 2 and 3 technical replicate 1", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 14 hpf", "GSM8327210", "GSM8327210: Reference experiment 14 hpf 24 hpf biological replicate 2 and 3 technical replicate 1; Danio rerio; OTHER", "GSM8327210 r1", "GSM8327210", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "ref_exp_brep2_3_trep1_S0_L003_R1_001.fastq.gz ref_exp_brep2_3_trep1_S0_L003_R2_001.fastq.gz ref_exp_brep2_3_trep1_S0_L003_R3_001.fastq.gz ref_exp_brep2_3_trep1_S0_L003_R4_001.fastq.gz", "fastq fastq fastq fastq", 10620575238.0, 116709618.0, "GSM8327210 r3", "0:61 1:8 2:8 3:14", "A:1967500221;C:1452601318;G:1411877165;T:2286950505;N:357489", 61, 8, 8, 14, 1967500221, 1452601318, 1411877165, 2286950505, 357489, "SRX24912661", "SRS21618596", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32776, "SRR29398915", "SRX24912661", "SRS21618596", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Reference experiment 14 hpf 24 hpf biological replicate 2 and 3 technical replicate 1", "GSM8327210", null, "source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 14 hpf|geo loc name:missing|collection date:missing", "Reference experiment 14 hpf 24 hpf biological replicate 2 and 3 technical replicate 1", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 14 hpf", "GSM8327210", "GSM8327210: Reference experiment 14 hpf 24 hpf biological replicate 2 and 3 technical replicate 1; Danio rerio; OTHER", "GSM8327210 r1", "GSM8327210", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "ref_exp_brep2_3_trep1_S0_L004_R1_001.fastq.gz ref_exp_brep2_3_trep1_S0_L004_R2_001.fastq.gz ref_exp_brep2_3_trep1_S0_L004_R3_001.fastq.gz ref_exp_brep2_3_trep1_S0_L004_R4_001.fastq.gz", "fastq fastq fastq fastq", 10584222740.0, 116310140.0, "GSM8327210 r4", "0:61 1:8 2:8 3:14", "A:1960717782;C:1446116581;G:1405414187;T:2282206014;N:463976", 61, 8, 8, 14, 1960717782, 1446116581, 1405414187, 2282206014, 463976, "SRX24912661", "SRS21618596", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32777, "SRR29398916", "SRX24912660", "SRS21618594", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Reference experiment 14 hpf 24 hpf biological replicate 1 technical replicate 1", "GSM8327209", null, "source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 14 hpf|geo loc name:missing|collection date:missing", "Reference experiment 14 hpf 24 hpf biological replicate 1 technical replicate 1", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 14 hpf", "GSM8327209", "GSM8327209: Reference experiment 14 hpf 24 hpf biological replicate 1 technical replicate 1; Danio rerio; OTHER", "GSM8327209 r1", "GSM8327209", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "ref_exp_brep1_trep1_S0_L001_R1_001.fastq.gz ref_exp_brep1_trep1_S0_L001_R2_001.fastq.gz ref_exp_brep1_trep1_S0_L001_R3_001.fastq.gz ref_exp_brep1_trep1_S0_L001_R4_001.fastq.gz", "fastq fastq fastq fastq", 12138066031.0, 133385341.0, "GSM8327209 r1", "0:61 1:8 2:8 3:14", "A:2284023815;C:1627684270;G:1586353604;T:2638230442;N:213670", 61, 8, 8, 14, 2284023815, 1627684270, 1586353604, 2638230442, 213670, "SRX24912660", "SRS21618594", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32778, "SRR29398917", "SRX24912660", "SRS21618594", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Reference experiment 14 hpf 24 hpf biological replicate 1 technical replicate 1", "GSM8327209", null, "source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 14 hpf|geo loc name:missing|collection date:missing", "Reference experiment 14 hpf 24 hpf biological replicate 1 technical replicate 1", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 14 hpf", "GSM8327209", "GSM8327209: Reference experiment 14 hpf 24 hpf biological replicate 1 technical replicate 1; Danio rerio; OTHER", "GSM8327209 r1", "GSM8327209", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "ref_exp_brep1_trep1_S0_L002_R1_001.fastq.gz ref_exp_brep1_trep1_S0_L002_R2_001.fastq.gz ref_exp_brep1_trep1_S0_L002_R3_001.fastq.gz ref_exp_brep1_trep1_S0_L002_R4_001.fastq.gz", "fastq fastq fastq fastq", 12243894572.0, 134548292.0, "GSM8327209 r2", "0:61 1:8 2:8 3:14", "A:2304388773;C:1639987412;G:1597880933;T:2665024548;N:164146", 61, 8, 8, 14, 2304388773, 1639987412, 1597880933, 2665024548, 164146, "SRX24912660", "SRS21618594", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32779, "SRR29398918", "SRX24912660", "SRS21618594", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Reference experiment 14 hpf 24 hpf biological replicate 1 technical replicate 1", "GSM8327209", null, "source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 14 hpf|geo loc name:missing|collection date:missing", "Reference experiment 14 hpf 24 hpf biological replicate 1 technical replicate 1", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 14 hpf", "GSM8327209", "GSM8327209: Reference experiment 14 hpf 24 hpf biological replicate 1 technical replicate 1; Danio rerio; OTHER", "GSM8327209 r1", "GSM8327209", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "ref_exp_brep1_trep1_S0_L003_R1_001.fastq.gz ref_exp_brep1_trep1_S0_L003_R2_001.fastq.gz ref_exp_brep1_trep1_S0_L003_R3_001.fastq.gz ref_exp_brep1_trep1_S0_L003_R4_001.fastq.gz", "fastq fastq fastq fastq", 12131674828.0, 133315108.0, "GSM8327209 r3", "0:61 1:8 2:8 3:14", "A:2285180187;C:1620399775;G:1589956627;T:2636408789;N:276210", 61, 8, 8, 14, 2285180187, 1620399775, 1589956627, 2636408789, 276210, "SRX24912660", "SRS21618594", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32780, "SRR29398919", "SRX24912660", "SRS21618594", "SRP513754", "PRJNA1123686", "Cell state transitions are decoupled from cell division during early embryo development [I]", "GSE269784", "Other", "As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: We arrested the cell cycle in zebrafish embryos at 6 hpf using two independent approaches: a chemical approach hydroxyurea and aphidicolin HUA and a genetic approach involving a loss of function mutation in the gene emi1 allele hi2648. We tracked differentiation dynamics using single cell RNA sequencing scRNA seq on embryos spanning 6\u201324 hpf for HUA treated and control embryos and at 24 hpf for emi1 mutant embryos. Overall we have collected multiple replicates for control embryos ABs at 6 8 10 14 18 21 hpf and 24 hpf for HUA treated at 8 10 14 hpf and 24 hpf and for emi1 mutants at 24 hpf. Details about the samples can be found in the supplementary file \"sample information.txt\"", null, "pubmed:37546736", null, "Reference experiment 14 hpf 24 hpf biological replicate 1 technical replicate 1", "GSM8327209", null, "source name:whole embryo|tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 14 hpf|geo loc name:missing|collection date:missing", "Reference experiment 14 hpf 24 hpf biological replicate 1 technical replicate 1", "Multiple samples are pooled for each sequencing run. Raw FASTQ were prepared from Illumina bcl files in a format compatible with the inDrops.py pipeline. Each nextseq run results in 16 FASTQ files 4 lanes x 4 reads per lane. For some pooled libraries sequencing was run twice to get more UMIs per cell and hence we have deposited 16x2 = 32 raw files for those sequencing samples. The illumina library indices of different samples in a run are provided in the meta data. These can be used to demultiplex the raw file into separate libraries. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity cell barcode and associated metadata for all cells time point treatment replicate annotated cell state total number of counts etc Library strategy: inDrops v3 scRNA seq", "whole embryo", "Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs \u2013 hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 \u00b5M concentration in 1% dimethyl sulfoxide DMSO in egg water. G2/M phase arrest was induced by crossing heterozygous emi1 wt/mut zebrafish to generate homozygous emi1 mut/mut embryos referred as hi2648 in the manuscript. Homozygous mutants with cell cycle arrest were screened at 24 hpf as they have very clear phenotype by this time \u2013 these embryos are smaller and have bent tails and the heterozygous mutants and wildtype are indistinguishable.", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", "AB wild type strains were used for all HUA perturbation experiments. Zebrafish mutant line hi2648 a mutant for the gene emi1 was kindly gifted by Dr. Jennifer Rhodes. Embryos were developed within the ambient temperature of Zebrafish development. Time of fertilization at 28.5 C was used to stage each clutch and this was confirmed using morphological features of the embryos. All zebrafish were housed in a facility overseen by the Harvard Medical Area Standing Committee on Animals our IACUC which performs regular inspections and under which we have an approved protocol for all animal procedures.", "tissue:whole embryo|treatment:1percent DMSO control and cell cycle arrest at 14 hpf", "GSM8327209", "GSM8327209: Reference experiment 14 hpf 24 hpf biological replicate 1 technical replicate 1; Danio rerio; OTHER", "GSM8327209 r1", "GSM8327209", "1", "Zebrafish embryos were dechorionated using 1mg/mL Pronase Sigma P5147 1G for 5 7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 hpf to 10 hpf embryos early time points and in 500 \u00b5L FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40\u00b5m cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP513754", null, null, "ref_exp_brep1_trep1_S0_L004_R1_001.fastq.gz ref_exp_brep1_trep1_S0_L004_R2_001.fastq.gz ref_exp_brep1_trep1_S0_L004_R3_001.fastq.gz ref_exp_brep1_trep1_S0_L004_R4_001.fastq.gz", "fastq fastq fastq fastq", 12104206569.0, 133013259.0, "GSM8327209 r4", "0:61 1:8 2:8 3:14", "A:2280991287;C:1619017132;G:1586873006;T:2626722786;N:204588", 61, 8, 8, 14, 2280991287, 1619017132, 1586873006, 2626722786, 204588, "SRX24912660", "SRS21618594", "SRA1898111", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-13", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [32781, "SRR29411985", "SRX24925451", "SRS21630866", "SRP513930", "PRJNA1124008", "Cell state transitions are decoupled from cell division during early embryo development [II]", "GSE269848", "Other", "Paper abstract: As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: Design of experiment for this particular dataset: Forty tails from zebrafish embryos at 24 38 hpf and 48 hpf were collected. Cells were dissociated from these tails and multi seq tags were added to the cells for each time point. Cells from all time points were pooled and single cell sequencing was performed using Indrops. Four gene expression GEX libraries were prepared for transcriptomic information and four multiseq tag libraries TAG were prepared for reading out the multiseq tags. We provide the raw data .fastq files processed data both raw counts.tsv.gz and filtered by total UMI counts filtered.h5ad files and the entire filtered data post removing doublets as all data.h5ad. We also provide counts for multi seq tags example TAG 1.counts.csv. Information about pooling libraries and library indices is provided in Pooling Samples.xlsx. Only 24 hpf data was used for Kukreja et. al. 2024 and hence the cell state information we provide in the all data.h5ad is only for 24 hpf.", null, "pubmed:37546736", null, "24 38 hpf and 48 hpf tails", "GSM8328864", null, "source name:embryo tail|tissue:embryo tail|geo loc name:missing|collection date:missing", "24 38 hpf and 48 hpf tails", "Reads were mapped onto the Zebrafish genome as described in Kukreja et al. 2024 manuscript. Multi seq barcodes for each sample were identified using a custom pipeline available here: https://github.com/AllonKleinLab/klunctions/. Barcode abundance was used to manually remove multiplet populations as well as assign cells to their appropriate sample. Data from only 24 hpf tails were used for the manuscript. We provide 8 FASTQ files 2 lanes x 4 reads per lane. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts for transcriptome tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Raw counts for multi seq tags csv.gz: cell barcode tag sequence and raw counts for all cells Supplementary files format and content: Filtered barcodes filtered.h5ad: cell barcode and associated metadata for all cells Library strategy: inDrops v3 scRNA seq", "embryo tail", null, "Forty zebrafish tail samples collected at 24 38 hpf and 48 hpf were dissected between the yolk ball and extension and dissociated according to a modified version of the protocol described by Bresciani et al. 2018. Briefly the tails were dissociated with a mixture of DNaseI 20\u00b5g/mL Collagenase/Dispase 8 mg/mL and 0.25% Trypsin EDTA at 30.5\u00b0C for 15 minutes. The proteases were quenched with DMEM + 10% FBS. Samples were washed and resuspend in PBS. Cells from each timepoint were hashed with a unique lipid modified oligo using Multi seq. The barcoded samples were subsequently pooled washed with 1% BSA + PBS and resuspended in 0.1% BSA + 18% Optiprep in PBS at a final concentration of 300 000 cells/mL. Single cell transcriptomes were captured using inDrops. NGS libraries were prepared by the Single cell Core at Harvard Medical School and sequenced using an Illumina NovaSeq kit.", null, "tissue:embryo tail", "GSM8328864", "GSM8328864: 24 38 hpf and 48 hpf tails; Danio rerio; OTHER", "GSM8328864 r1", "GSM8328864", "1", "Forty zebrafish tail samples collected at 24 38 hpf and 48 hpf were dissected between the yolk ball and extension and dissociated according to a modified version of the protocol described by Bresciani et al. 2018. Briefly the tails were dissociated with a mixture of DNaseI 20\u00b5g/mL Collagenase/Dispase 8 mg/mL and 0.25% Trypsin EDTA at 30.5\u00b0C for 15 minutes. The proteases were quenched with DMEM + 10% FBS. Samples were washed and resuspend in PBS. Cells from each timepoint were hashed with a unique lipid modified oligo using Multi seq. The barcoded samples were subsequently pooled washed with 1% BSA + PBS and resuspended in 0.1% BSA + 18% Optiprep in PBS at a final concentration of 300 000 cells/mL. Single cell transcriptomes were captured using inDrops. NGS libraries were prepared by the Single cell Core at Harvard Medical School and sequenced using an Illumina NovaSeq kit.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP513930", null, null, "Undetermined_S0_L002_R1_001.fastq.gz Undetermined_S0_L002_R2_001.fastq.gz Undetermined_S0_L002_R3_001.fastq.gz Undetermined_S0_L002_R4_001.fastq.gz", "fastq fastq fastq fastq", 58603546956.0, 505202991.0, "GSM8328864 r1", "0:86 1:8 2:8 3:14", "A:13191204330;C:9162028793;G:9431065581;T:11662090182;N:1068340", 86, 8, 8, 14, 13191204330, 9162028793, 9431065581, 11662090182, 1068340, "SRX24925451", "SRS21630866", "SRA1899240", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "novaseq_era", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-14", "Multi-stage", "Embryo", "Tail", "Multi-system"], [32782, "SRR29411986", "SRX24925451", "SRS21630866", "SRP513930", "PRJNA1124008", "Cell state transitions are decoupled from cell division during early embryo development [II]", "GSE269848", "Other", "Paper abstract: As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: Design of experiment for this particular dataset: Forty tails from zebrafish embryos at 24 38 hpf and 48 hpf were collected. Cells were dissociated from these tails and multi seq tags were added to the cells for each time point. Cells from all time points were pooled and single cell sequencing was performed using Indrops. Four gene expression GEX libraries were prepared for transcriptomic information and four multiseq tag libraries TAG were prepared for reading out the multiseq tags. We provide the raw data .fastq files processed data both raw counts.tsv.gz and filtered by total UMI counts filtered.h5ad files and the entire filtered data post removing doublets as all data.h5ad. We also provide counts for multi seq tags example TAG 1.counts.csv. Information about pooling libraries and library indices is provided in Pooling Samples.xlsx. Only 24 hpf data was used for Kukreja et. al. 2024 and hence the cell state information we provide in the all data.h5ad is only for 24 hpf.", null, "pubmed:37546736", null, "24 38 hpf and 48 hpf tails", "GSM8328864", null, "source name:embryo tail|tissue:embryo tail|geo loc name:missing|collection date:missing", "24 38 hpf and 48 hpf tails", "Reads were mapped onto the Zebrafish genome as described in Kukreja et al. 2024 manuscript. Multi seq barcodes for each sample were identified using a custom pipeline available here: https://github.com/AllonKleinLab/klunctions/. Barcode abundance was used to manually remove multiplet populations as well as assign cells to their appropriate sample. Data from only 24 hpf tails were used for the manuscript. We provide 8 FASTQ files 2 lanes x 4 reads per lane. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section \"Single cell RNA seq Data preprocessing\" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts for transcriptome tsv.gz: cell barcode gene names and raw counts for all cells Supplementary files format and content: Raw counts for multi seq tags csv.gz: cell barcode tag sequence and raw counts for all cells Supplementary files format and content: Filtered barcodes filtered.h5ad: cell barcode and associated metadata for all cells Library strategy: inDrops v3 scRNA seq", "embryo tail", null, "Forty zebrafish tail samples collected at 24 38 hpf and 48 hpf were dissected between the yolk ball and extension and dissociated according to a modified version of the protocol described by Bresciani et al. 2018. Briefly the tails were dissociated with a mixture of DNaseI 20\u00b5g/mL Collagenase/Dispase 8 mg/mL and 0.25% Trypsin EDTA at 30.5\u00b0C for 15 minutes. The proteases were quenched with DMEM + 10% FBS. Samples were washed and resuspend in PBS. Cells from each timepoint were hashed with a unique lipid modified oligo using Multi seq. The barcoded samples were subsequently pooled washed with 1% BSA + PBS and resuspended in 0.1% BSA + 18% Optiprep in PBS at a final concentration of 300 000 cells/mL. Single cell transcriptomes were captured using inDrops. NGS libraries were prepared by the Single cell Core at Harvard Medical School and sequenced using an Illumina NovaSeq kit.", null, "tissue:embryo tail", "GSM8328864", "GSM8328864: 24 38 hpf and 48 hpf tails; Danio rerio; OTHER", "GSM8328864 r1", "GSM8328864", "1", "Forty zebrafish tail samples collected at 24 38 hpf and 48 hpf were dissected between the yolk ball and extension and dissociated according to a modified version of the protocol described by Bresciani et al. 2018. Briefly the tails were dissociated with a mixture of DNaseI 20\u00b5g/mL Collagenase/Dispase 8 mg/mL and 0.25% Trypsin EDTA at 30.5\u00b0C for 15 minutes. The proteases were quenched with DMEM + 10% FBS. Samples were washed and resuspend in PBS. Cells from each timepoint were hashed with a unique lipid modified oligo using Multi seq. The barcoded samples were subsequently pooled washed with 1% BSA + PBS and resuspended in 0.1% BSA + 18% Optiprep in PBS at a final concentration of 300 000 cells/mL. Single cell transcriptomes were captured using inDrops. NGS libraries were prepared by the Single cell Core at Harvard Medical School and sequenced using an Illumina NovaSeq kit.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP513930", null, null, "Undetermined_S0_L001_R1_001.fastq.gz Undetermined_S0_L001_R2_001.fastq.gz Undetermined_S0_L001_R3_001.fastq.gz Undetermined_S0_L001_R4_001.fastq.gz", "fastq fastq fastq fastq", 44072422880.0, 379934680.0, "GSM8328864 r2", "0:86 1:8 2:8 3:14", "A:9919784228;C:6894965731;G:7094749745;T:8764100624;N:782152", 86, 8, 8, 14, 9919784228, 6894965731, 7094749745, 8764100624, 782152, "SRX24925451", "SRS21630866", "SRA1899240", "Harvard University", "Harvard University", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "novaseq_era", "unknown", "other", "trueseq", "sc", "single_cell_droplet", "indrops", null, "United States", "2024-06-14", "Multi-stage", "Embryo", "Tail", "Multi-system"], [47758, "SRR6890914", "SRX3841335", "SRS3087495", "SRP136369", "PRJNA445487", "Systematic mapping of cell state trajectories cell lineage and perturbations in the zebrafish embryo using single cell transcriptomics", "GSE112294", "Transcriptome Analysis", "High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease. Here we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach we mapped a cell state landscape that describes axis patterning germ layer formation and organogenesis. We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach \u201cTracerSeq\u201d for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.", null, "pubmed:29700229", null, "CRISPR Targeted Embryo tyrosinase C", "GSM3067206", null, "tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:tyrosinase targeted Cas9", "CRISPR Targeted Embryo tyrosinase C", "inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al. Nature Protocols 2017 and https://github.com/indrops/. Each sequencing spot is associated with a single biological read and 1 3 technical reads depending on the specific inDrops library preparation chemistry used. FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read. DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read. DEW101 208 used V3 chemistry in which read1 is the biological read read2 carries the first half of the cell barcode read3 carries the library index and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 \"Identify abundant barcodes\" see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1 parameters: n 1 l 15 e 200 m 200 best strata a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads. This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table. UMI counts were adjusted by a total counts normalization. For TracerSeq embryos inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above. These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt. An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv. A table of annotations for each ClusterID. Convert DEW to SRR csv. A table for converting between DEW and NCBI library names. CellTracerCounts csv. A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.", "Zebrafish Embryo Dissociated Cells", null, "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", null, "strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:tyrosinase targeted Cas9", "GSM3067206", "GSM3067206: CRISPR Targeted Embryo tyrosinase C; Danio rerio; RNA Seq", "GSM3067206", null, "1", "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", "GEO Accession:GSM3067206", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP136369", null, "loader:fastq load.py|options: appendBCtoName platform=Illumina", "DEW203.fastq.gz", "fastq", 1465038283.0, 25330646.0, "GSM3067206 r1", "0:57.84", "A:399530311;C:292071189;G:290193165;T:483241417;N:2201", 57, null, null, null, 399530311, 292071189, 290193165, 483241417, 2201, "SRX3841335", "SRS3087495", "SRA672434", "GEO", "Systems Biology, Harvard Medical School", 1, 0.88338, null, 0.13639, null, 0.78295, null, 0.48646, null, 61, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2018-03-23", "Segmentation", "Embryo", "Embryo Imprecise", "All anatomical structures"], [47759, "SRR6890915", "SRX3841335", "SRS3087495", "SRP136369", "PRJNA445487", "Systematic mapping of cell state trajectories cell lineage and perturbations in the zebrafish embryo using single cell transcriptomics", "GSE112294", "Transcriptome Analysis", "High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease. Here we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach we mapped a cell state landscape that describes axis patterning germ layer formation and organogenesis. We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach \u201cTracerSeq\u201d for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.", null, "pubmed:29700229", null, "CRISPR Targeted Embryo tyrosinase C", "GSM3067206", null, "tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:tyrosinase targeted Cas9", "CRISPR Targeted Embryo tyrosinase C", "inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al. Nature Protocols 2017 and https://github.com/indrops/. Each sequencing spot is associated with a single biological read and 1 3 technical reads depending on the specific inDrops library preparation chemistry used. FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read. DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read. DEW101 208 used V3 chemistry in which read1 is the biological read read2 carries the first half of the cell barcode read3 carries the library index and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 \"Identify abundant barcodes\" see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1 parameters: n 1 l 15 e 200 m 200 best strata a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads. This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table. UMI counts were adjusted by a total counts normalization. For TracerSeq embryos inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above. These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt. An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv. A table of annotations for each ClusterID. Convert DEW to SRR csv. A table for converting between DEW and NCBI library names. CellTracerCounts csv. A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.", "Zebrafish Embryo Dissociated Cells", null, "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", null, "strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:tyrosinase targeted Cas9", "GSM3067206", "GSM3067206: CRISPR Targeted Embryo tyrosinase C; Danio rerio; RNA Seq", "GSM3067206", null, "1", "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", "GEO Accession:GSM3067206", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP136369", null, "loader:fastq load.py|options: appendBCtoName platform=Illumina", "DEW204.fastq.gz", "fastq", 1462027529.0, 25327645.0, "GSM3067206 r2", "0:57.72", "A:396943012;C:291806975;G:292100327;T:481174977;N:2238", 57, null, null, null, 396943012, 291806975, 292100327, 481174977, 2238, "SRX3841335", "SRS3087495", "SRA672434", "GEO", "Systems Biology, Harvard Medical School", 1, 0.88375, null, 0.13748, null, 0.78589, null, 0.49104, null, 60, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2018-03-23", "Segmentation", "Embryo", "Embryo Imprecise", "All anatomical structures"], [47760, "SRR6890916", "SRX3841335", "SRS3087495", "SRP136369", "PRJNA445487", "Systematic mapping of cell state trajectories cell lineage and perturbations in the zebrafish embryo using single cell transcriptomics", "GSE112294", "Transcriptome Analysis", "High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease. Here we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach we mapped a cell state landscape that describes axis patterning germ layer formation and organogenesis. We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach \u201cTracerSeq\u201d for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.", null, "pubmed:29700229", null, "CRISPR Targeted Embryo tyrosinase C", "GSM3067206", null, "tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:tyrosinase targeted Cas9", "CRISPR Targeted Embryo tyrosinase C", "inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al. Nature Protocols 2017 and https://github.com/indrops/. Each sequencing spot is associated with a single biological read and 1 3 technical reads depending on the specific inDrops library preparation chemistry used. FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read. DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read. DEW101 208 used V3 chemistry in which read1 is the biological read read2 carries the first half of the cell barcode read3 carries the library index and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 \"Identify abundant barcodes\" see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1 parameters: n 1 l 15 e 200 m 200 best strata a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads. This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table. UMI counts were adjusted by a total counts normalization. For TracerSeq embryos inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above. These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt. An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv. A table of annotations for each ClusterID. Convert DEW to SRR csv. A table for converting between DEW and NCBI library names. CellTracerCounts csv. A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.", "Zebrafish Embryo Dissociated Cells", null, "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", null, "strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:tyrosinase targeted Cas9", "GSM3067206", "GSM3067206: CRISPR Targeted Embryo tyrosinase C; Danio rerio; RNA Seq", "GSM3067206", null, "1", "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", "GEO Accession:GSM3067206", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP136369", null, "loader:fastq load.py|options: appendBCtoName platform=Illumina", "DEW205.fastq.gz", "fastq", 1089879965.0, 19024780.0, "GSM3067206 r3", "0:57.29", "A:299577336;C:213749796;G:214826324;T:361724880;N:1629", 57, null, null, null, 299577336, 213749796, 214826324, 361724880, 1629, "SRX3841335", "SRS3087495", "SRA672434", "GEO", "Systems Biology, Harvard Medical School", 1, 0.88076, null, 0.13757, null, 0.78324, null, 0.4895, null, 61, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2018-03-23", "Segmentation", "Embryo", "Embryo Imprecise", "All anatomical structures"], [47761, "SRR6890912", "SRX3841334", "SRS3087494", "SRP136369", "PRJNA445487", "Systematic mapping of cell state trajectories cell lineage and perturbations in the zebrafish embryo using single cell transcriptomics", "GSE112294", "Transcriptome Analysis", "High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease. Here we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach we mapped a cell state landscape that describes axis patterning germ layer formation and organogenesis. We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach \u201cTracerSeq\u201d for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.", null, "pubmed:29700229", null, "CRISPR Targeted Embryo tyrosinase B", "GSM3067205", null, "tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:tyrosinase targeted Cas9", "CRISPR Targeted Embryo tyrosinase B", "inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al. Nature Protocols 2017 and https://github.com/indrops/. Each sequencing spot is associated with a single biological read and 1 3 technical reads depending on the specific inDrops library preparation chemistry used. FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read. DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read. DEW101 208 used V3 chemistry in which read1 is the biological read read2 carries the first half of the cell barcode read3 carries the library index and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 \"Identify abundant barcodes\" see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1 parameters: n 1 l 15 e 200 m 200 best strata a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads. This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table. UMI counts were adjusted by a total counts normalization. For TracerSeq embryos inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above. These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt. An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv. A table of annotations for each ClusterID. Convert DEW to SRR csv. A table for converting between DEW and NCBI library names. CellTracerCounts csv. A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.", "Zebrafish Embryo Dissociated Cells", null, "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", null, "strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:tyrosinase targeted Cas9", "GSM3067205", "GSM3067205: CRISPR Targeted Embryo tyrosinase B; Danio rerio; RNA Seq", "GSM3067205", null, "1", "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", "GEO Accession:GSM3067205", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP136369", null, "loader:fastq load.py|options: appendBCtoName platform=Illumina", "DEW198.fastq.gz", "fastq", 1750603340.0, 30262674.0, "GSM3067205 r1", "0:57.85", "A:476772061;C:348089294;G:341349666;T:584389613;N:2706", 57, null, null, null, 476772061, 348089294, 341349666, 584389613, 2706, "SRX3841334", "SRS3087494", "SRA672434", "GEO", "Systems Biology, Harvard Medical School", 1, 0.88041, null, 0.13852, null, 0.79052, null, 0.50356, null, 61, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2018-03-23", "Segmentation", "Embryo", "Embryo Imprecise", "All anatomical structures"], [47762, "SRR6890913", "SRX3841334", "SRS3087494", "SRP136369", "PRJNA445487", "Systematic mapping of cell state trajectories cell lineage and perturbations in the zebrafish embryo using single cell transcriptomics", "GSE112294", "Transcriptome Analysis", "High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease. Here we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach we mapped a cell state landscape that describes axis patterning germ layer formation and organogenesis. We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach \u201cTracerSeq\u201d for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.", null, "pubmed:29700229", null, "CRISPR Targeted Embryo tyrosinase B", "GSM3067205", null, "tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:tyrosinase targeted Cas9", "CRISPR Targeted Embryo tyrosinase B", "inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al. Nature Protocols 2017 and https://github.com/indrops/. Each sequencing spot is associated with a single biological read and 1 3 technical reads depending on the specific inDrops library preparation chemistry used. FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read. DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read. DEW101 208 used V3 chemistry in which read1 is the biological read read2 carries the first half of the cell barcode read3 carries the library index and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 \"Identify abundant barcodes\" see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1 parameters: n 1 l 15 e 200 m 200 best strata a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads. This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table. UMI counts were adjusted by a total counts normalization. For TracerSeq embryos inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above. These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt. An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv. A table of annotations for each ClusterID. Convert DEW to SRR csv. A table for converting between DEW and NCBI library names. CellTracerCounts csv. A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.", "Zebrafish Embryo Dissociated Cells", null, "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", null, "strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:tyrosinase targeted Cas9", "GSM3067205", "GSM3067205: CRISPR Targeted Embryo tyrosinase B; Danio rerio; RNA Seq", "GSM3067205", null, "1", "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", "GEO Accession:GSM3067205", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP136369", null, "loader:fastq load.py|options: appendBCtoName platform=Illumina", "DEW199.fastq.gz", "fastq", 1719747339.0, 29740919.0, "GSM3067205 r2", "0:57.82", "A:465181744;C:338602328;G:336843629;T:579117056;N:2582", 57, null, null, null, 465181744, 338602328, 336843629, 579117056, 2582, "SRX3841334", "SRS3087494", "SRA672434", "GEO", "Systems Biology, Harvard Medical School", 1, 0.89022, null, 0.13973, null, 0.79046, null, 0.47161, null, 60, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2018-03-23", "Segmentation", "Embryo", "Embryo Imprecise", "All anatomical structures"], [47763, "SRR6890910", "SRX3841333", "SRS3087493", "SRP136369", "PRJNA445487", "Systematic mapping of cell state trajectories cell lineage and perturbations in the zebrafish embryo using single cell transcriptomics", "GSE112294", "Transcriptome Analysis", "High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease. Here we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach we mapped a cell state landscape that describes axis patterning germ layer formation and organogenesis. We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach \u201cTracerSeq\u201d for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.", null, "pubmed:29700229", null, "CRISPR Targeted Embryo tyrosinase A", "GSM3067204", null, "tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:tyrosinase targeted Cas9", "CRISPR Targeted Embryo tyrosinase A", "inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al. Nature Protocols 2017 and https://github.com/indrops/. Each sequencing spot is associated with a single biological read and 1 3 technical reads depending on the specific inDrops library preparation chemistry used. FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read. DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read. DEW101 208 used V3 chemistry in which read1 is the biological read read2 carries the first half of the cell barcode read3 carries the library index and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 \"Identify abundant barcodes\" see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1 parameters: n 1 l 15 e 200 m 200 best strata a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads. This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table. UMI counts were adjusted by a total counts normalization. For TracerSeq embryos inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above. These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt. An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv. A table of annotations for each ClusterID. Convert DEW to SRR csv. A table for converting between DEW and NCBI library names. CellTracerCounts csv. A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.", "Zebrafish Embryo Dissociated Cells", null, "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", null, "strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:tyrosinase targeted Cas9", "GSM3067204", "GSM3067204: CRISPR Targeted Embryo tyrosinase A; Danio rerio; RNA Seq", "GSM3067204", null, "1", "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", "GEO Accession:GSM3067204", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP136369", null, "loader:fastq load.py|options: appendBCtoName platform=Illumina", "DEW191.fastq.gz", "fastq", 1430918940.0, 24757119.0, "GSM3067204 r1", "0:57.80", "A:386848505;C:283215419;G:279611431;T:481241360;N:2225", 57, null, null, null, 386848505, 283215419, 279611431, 481241360, 2225, "SRX3841333", "SRS3087493", "SRA672434", "GEO", "Systems Biology, Harvard Medical School", 1, 0.88097, null, 0.1317, null, 0.79722, null, 0.50104, null, 20, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2018-03-23", "Segmentation", "Embryo", "Embryo Imprecise", "All anatomical structures"], [47764, "SRR6890911", "SRX3841333", "SRS3087493", "SRP136369", "PRJNA445487", "Systematic mapping of cell state trajectories cell lineage and perturbations in the zebrafish embryo using single cell transcriptomics", "GSE112294", "Transcriptome Analysis", "High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease. Here we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach we mapped a cell state landscape that describes axis patterning germ layer formation and organogenesis. We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach \u201cTracerSeq\u201d for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.", null, "pubmed:29700229", null, "CRISPR Targeted Embryo tyrosinase A", "GSM3067204", null, "tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:tyrosinase targeted Cas9", "CRISPR Targeted Embryo tyrosinase A", "inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al. Nature Protocols 2017 and https://github.com/indrops/. Each sequencing spot is associated with a single biological read and 1 3 technical reads depending on the specific inDrops library preparation chemistry used. FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read. DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read. DEW101 208 used V3 chemistry in which read1 is the biological read read2 carries the first half of the cell barcode read3 carries the library index and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 \"Identify abundant barcodes\" see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1 parameters: n 1 l 15 e 200 m 200 best strata a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads. This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table. UMI counts were adjusted by a total counts normalization. For TracerSeq embryos inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above. These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt. An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv. A table of annotations for each ClusterID. Convert DEW to SRR csv. A table for converting between DEW and NCBI library names. CellTracerCounts csv. A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.", "Zebrafish Embryo Dissociated Cells", null, "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", null, "strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:tyrosinase targeted Cas9", "GSM3067204", "GSM3067204: CRISPR Targeted Embryo tyrosinase A; Danio rerio; RNA Seq", "GSM3067204", null, "1", "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", "GEO Accession:GSM3067204", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP136369", null, "loader:fastq load.py|options: appendBCtoName platform=Illumina", "DEW192.fastq.gz", "fastq", 1460183677.0, 25279012.0, "GSM3067204 r2", "0:57.76", "A:393214592;C:288843309;G:287072676;T:491050801;N:2299", 57, null, null, null, 393214592, 288843309, 287072676, 491050801, 2299, "SRX3841333", "SRS3087493", "SRA672434", "GEO", "Systems Biology, Harvard Medical School", 1, 0.88188, null, 0.13315, null, 0.79878, null, 0.47807, null, 61, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2018-03-23", "Segmentation", "Embryo", "Embryo Imprecise", "All anatomical structures"], [47765, "SRR6890907", "SRX3841332", "SRS3087492", "SRP136369", "PRJNA445487", "Systematic mapping of cell state trajectories cell lineage and perturbations in the zebrafish embryo using single cell transcriptomics", "GSE112294", "Transcriptome Analysis", "High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease. Here we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach we mapped a cell state landscape that describes axis patterning germ layer formation and organogenesis. We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach \u201cTracerSeq\u201d for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.", null, "pubmed:29700229", null, "CRISPR Targeted Embryo chordin C", "GSM3067203", null, "tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:chordin targeted Cas9", "CRISPR Targeted Embryo chordin C", "inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al. Nature Protocols 2017 and https://github.com/indrops/. Each sequencing spot is associated with a single biological read and 1 3 technical reads depending on the specific inDrops library preparation chemistry used. FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read. DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read. DEW101 208 used V3 chemistry in which read1 is the biological read read2 carries the first half of the cell barcode read3 carries the library index and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 \"Identify abundant barcodes\" see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1 parameters: n 1 l 15 e 200 m 200 best strata a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads. This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table. UMI counts were adjusted by a total counts normalization. For TracerSeq embryos inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above. These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt. An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv. A table of annotations for each ClusterID. Convert DEW to SRR csv. A table for converting between DEW and NCBI library names. CellTracerCounts csv. A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.", "Zebrafish Embryo Dissociated Cells", null, "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", null, "strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:chordin targeted Cas9", "GSM3067203", "GSM3067203: CRISPR Targeted Embryo chordin C; Danio rerio; RNA Seq", "GSM3067203", null, "1", "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", "GEO Accession:GSM3067203", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP136369", null, "loader:fastq load.py|options: appendBCtoName platform=Illumina", "DEW206.fastq.gz", "fastq", 1176220272.0, 20354110.0, "GSM3067203 r1", "0:57.79", "A:321130163;C:233656608;G:233960194;T:387471545;N:1762", 57, null, null, null, 321130163, 233656608, 233960194, 387471545, 1762, "SRX3841332", "SRS3087492", "SRA672434", "GEO", "Systems Biology, Harvard Medical School", 1, 0.88335, null, 0.15046, null, 0.78559, null, 0.52197, null, 61, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2018-03-23", "Segmentation", "Embryo", "Embryo Imprecise", "All anatomical structures"], [47766, "SRR6890908", "SRX3841332", "SRS3087492", "SRP136369", "PRJNA445487", "Systematic mapping of cell state trajectories cell lineage and perturbations in the zebrafish embryo using single cell transcriptomics", "GSE112294", "Transcriptome Analysis", "High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease. Here we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach we mapped a cell state landscape that describes axis patterning germ layer formation and organogenesis. We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach \u201cTracerSeq\u201d for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.", null, "pubmed:29700229", null, "CRISPR Targeted Embryo chordin C", "GSM3067203", null, "tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:chordin targeted Cas9", "CRISPR Targeted Embryo chordin C", "inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al. Nature Protocols 2017 and https://github.com/indrops/. Each sequencing spot is associated with a single biological read and 1 3 technical reads depending on the specific inDrops library preparation chemistry used. FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read. DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read. DEW101 208 used V3 chemistry in which read1 is the biological read read2 carries the first half of the cell barcode read3 carries the library index and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 \"Identify abundant barcodes\" see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1 parameters: n 1 l 15 e 200 m 200 best strata a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads. This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table. UMI counts were adjusted by a total counts normalization. For TracerSeq embryos inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above. These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt. An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv. A table of annotations for each ClusterID. Convert DEW to SRR csv. A table for converting between DEW and NCBI library names. CellTracerCounts csv. A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.", "Zebrafish Embryo Dissociated Cells", null, "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", null, "strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:chordin targeted Cas9", "GSM3067203", "GSM3067203: CRISPR Targeted Embryo chordin C; Danio rerio; RNA Seq", "GSM3067203", null, "1", "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", "GEO Accession:GSM3067203", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP136369", null, "loader:fastq load.py|options: appendBCtoName platform=Illumina", "DEW207.fastq.gz", "fastq", 1094042604.0, 18956943.0, "GSM3067203 r2", "0:57.71", "A:299520746;C:217679611;G:217383022;T:359457558;N:1667", 57, null, null, null, 299520746, 217679611, 217383022, 359457558, 1667, "SRX3841332", "SRS3087492", "SRA672434", "GEO", "Systems Biology, Harvard Medical School", 1, 0.87731, null, 0.14126, null, 0.78455, null, 0.51361, null, 61, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2018-03-23", "Segmentation", "Embryo", "Embryo Imprecise", "All anatomical structures"], [47767, "SRR6890909", "SRX3841332", "SRS3087492", "SRP136369", "PRJNA445487", "Systematic mapping of cell state trajectories cell lineage and perturbations in the zebrafish embryo using single cell transcriptomics", "GSE112294", "Transcriptome Analysis", "High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease. Here we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach we mapped a cell state landscape that describes axis patterning germ layer formation and organogenesis. We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach \u201cTracerSeq\u201d for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.", null, "pubmed:29700229", null, "CRISPR Targeted Embryo chordin C", "GSM3067203", null, "tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:chordin targeted Cas9", "CRISPR Targeted Embryo chordin C", "inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al. Nature Protocols 2017 and https://github.com/indrops/. Each sequencing spot is associated with a single biological read and 1 3 technical reads depending on the specific inDrops library preparation chemistry used. FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read. DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read. DEW101 208 used V3 chemistry in which read1 is the biological read read2 carries the first half of the cell barcode read3 carries the library index and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 \"Identify abundant barcodes\" see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1 parameters: n 1 l 15 e 200 m 200 best strata a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads. This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table. UMI counts were adjusted by a total counts normalization. For TracerSeq embryos inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above. These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt. An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv. A table of annotations for each ClusterID. Convert DEW to SRR csv. A table for converting between DEW and NCBI library names. CellTracerCounts csv. A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.", "Zebrafish Embryo Dissociated Cells", null, "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", null, "strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:chordin targeted Cas9", "GSM3067203", "GSM3067203: CRISPR Targeted Embryo chordin C; Danio rerio; RNA Seq", "GSM3067203", null, "1", "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", "GEO Accession:GSM3067203", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP136369", null, "loader:fastq load.py|options: appendBCtoName platform=Illumina", "DEW208.fastq.gz", "fastq", 1096168422.0, 18997947.0, "GSM3067203 r3", "0:57.70", "A:299037227;C:219614732;G:219564739;T:357950083;N:1641", 57, null, null, null, 299037227, 219614732, 219564739, 357950083, 1641, "SRX3841332", "SRS3087492", "SRA672434", "GEO", "Systems Biology, Harvard Medical School", 1, 0.87903, null, 0.1438, null, 0.78652, null, 0.50674, null, 61, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2018-03-23", "Segmentation", "Embryo", "Embryo Imprecise", "All anatomical structures"], [47768, "SRR6890904", "SRX3841331", "SRS3087491", "SRP136369", "PRJNA445487", "Systematic mapping of cell state trajectories cell lineage and perturbations in the zebrafish embryo using single cell transcriptomics", "GSE112294", "Transcriptome Analysis", "High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease. Here we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach we mapped a cell state landscape that describes axis patterning germ layer formation and organogenesis. We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach \u201cTracerSeq\u201d for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.", null, "pubmed:29700229", null, "CRISPR Targeted Embryo chordin B", "GSM3067202", null, "tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:chordin targeted Cas9", "CRISPR Targeted Embryo chordin B", "inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al. Nature Protocols 2017 and https://github.com/indrops/. Each sequencing spot is associated with a single biological read and 1 3 technical reads depending on the specific inDrops library preparation chemistry used. FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read. DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read. DEW101 208 used V3 chemistry in which read1 is the biological read read2 carries the first half of the cell barcode read3 carries the library index and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 \"Identify abundant barcodes\" see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1 parameters: n 1 l 15 e 200 m 200 best strata a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads. This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table. UMI counts were adjusted by a total counts normalization. For TracerSeq embryos inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above. These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt. An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv. A table of annotations for each ClusterID. Convert DEW to SRR csv. A table for converting between DEW and NCBI library names. CellTracerCounts csv. A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.", "Zebrafish Embryo Dissociated Cells", null, "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", null, "strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:chordin targeted Cas9", "GSM3067202", "GSM3067202: CRISPR Targeted Embryo chordin B; Danio rerio; RNA Seq", "GSM3067202", null, "1", "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", "GEO Accession:GSM3067202", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP136369", null, "loader:fastq load.py|options: appendBCtoName platform=Illumina", "DEW200.fastq.gz", "fastq", 1515827613.0, 26281497.0, "GSM3067202 r1", "0:57.68", "A:416122854;C:299949702;G:299356908;T:500395913;N:2236", 57, null, null, null, 416122854, 299949702, 299356908, 500395913, 2236, "SRX3841331", "SRS3087491", "SRA672434", "GEO", "Systems Biology, Harvard Medical School", 1, 0.87584, null, 0.15434, null, 0.78522, null, 0.51754, null, 61, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2018-03-23", "Segmentation", "Embryo", "Embryo Imprecise", "All anatomical structures"], [47769, "SRR6890905", "SRX3841331", "SRS3087491", "SRP136369", "PRJNA445487", "Systematic mapping of cell state trajectories cell lineage and perturbations in the zebrafish embryo using single cell transcriptomics", "GSE112294", "Transcriptome Analysis", "High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease. Here we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach we mapped a cell state landscape that describes axis patterning germ layer formation and organogenesis. We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach \u201cTracerSeq\u201d for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.", null, "pubmed:29700229", null, "CRISPR Targeted Embryo chordin B", "GSM3067202", null, "tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:chordin targeted Cas9", "CRISPR Targeted Embryo chordin B", "inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al. Nature Protocols 2017 and https://github.com/indrops/. Each sequencing spot is associated with a single biological read and 1 3 technical reads depending on the specific inDrops library preparation chemistry used. FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read. DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read. DEW101 208 used V3 chemistry in which read1 is the biological read read2 carries the first half of the cell barcode read3 carries the library index and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 \"Identify abundant barcodes\" see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1 parameters: n 1 l 15 e 200 m 200 best strata a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads. This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table. UMI counts were adjusted by a total counts normalization. For TracerSeq embryos inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above. These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt. An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv. A table of annotations for each ClusterID. Convert DEW to SRR csv. A table for converting between DEW and NCBI library names. CellTracerCounts csv. A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.", "Zebrafish Embryo Dissociated Cells", null, "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", null, "strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:chordin targeted Cas9", "GSM3067202", "GSM3067202: CRISPR Targeted Embryo chordin B; Danio rerio; RNA Seq", "GSM3067202", null, "1", "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", "GEO Accession:GSM3067202", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP136369", null, "loader:fastq load.py|options: appendBCtoName platform=Illumina", "DEW201.fastq.gz", "fastq", 1486290397.0, 25698034.0, "GSM3067202 r2", "0:57.84", "A:405024414;C:294656621;G:294652070;T:491954998;N:2294", 57, null, null, null, 405024414, 294656621, 294652070, 491954998, 2294, "SRX3841331", "SRS3087491", "SRA672434", "GEO", "Systems Biology, Harvard Medical School", 1, 0.88539, null, 0.1652, null, 0.78922, null, 0.51537, null, 61, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2018-03-23", "Segmentation", "Embryo", "Embryo Imprecise", "All anatomical structures"], [47770, "SRR6890906", "SRX3841331", "SRS3087491", "SRP136369", "PRJNA445487", "Systematic mapping of cell state trajectories cell lineage and perturbations in the zebrafish embryo using single cell transcriptomics", "GSE112294", "Transcriptome Analysis", "High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease. Here we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach we mapped a cell state landscape that describes axis patterning germ layer formation and organogenesis. We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach \u201cTracerSeq\u201d for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.", null, "pubmed:29700229", null, "CRISPR Targeted Embryo chordin B", "GSM3067202", null, "tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:chordin targeted Cas9", "CRISPR Targeted Embryo chordin B", "inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al. Nature Protocols 2017 and https://github.com/indrops/. Each sequencing spot is associated with a single biological read and 1 3 technical reads depending on the specific inDrops library preparation chemistry used. FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read. DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read. DEW101 208 used V3 chemistry in which read1 is the biological read read2 carries the first half of the cell barcode read3 carries the library index and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 \"Identify abundant barcodes\" see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1 parameters: n 1 l 15 e 200 m 200 best strata a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads. This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table. UMI counts were adjusted by a total counts normalization. For TracerSeq embryos inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above. These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt. An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv. A table of annotations for each ClusterID. Convert DEW to SRR csv. A table for converting between DEW and NCBI library names. CellTracerCounts csv. A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.", "Zebrafish Embryo Dissociated Cells", null, "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", null, "strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:chordin targeted Cas9", "GSM3067202", "GSM3067202: CRISPR Targeted Embryo chordin B; Danio rerio; RNA Seq", "GSM3067202", null, "1", "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", "GEO Accession:GSM3067202", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP136369", null, "loader:fastq load.py|options: appendBCtoName platform=Illumina", "DEW202.fastq.gz", "fastq", 1381539779.0, 23983713.0, "GSM3067202 r3", "0:57.60", "A:376598971;C:275317367;G:275659505;T:453961857;N:2079", 57, null, null, null, 376598971, 275317367, 275659505, 453961857, 2079, "SRX3841331", "SRS3087491", "SRA672434", "GEO", "Systems Biology, Harvard Medical School", 1, 0.88032, null, 0.15709, null, 0.79017, null, 0.5072, null, 58, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2018-03-23", "Segmentation", "Embryo", "Embryo Imprecise", "All anatomical structures"], [47771, "SRR6890899", "SRX3841330", "SRS3087490", "SRP136369", "PRJNA445487", "Systematic mapping of cell state trajectories cell lineage and perturbations in the zebrafish embryo using single cell transcriptomics", "GSE112294", "Transcriptome Analysis", "High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease. Here we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach we mapped a cell state landscape that describes axis patterning germ layer formation and organogenesis. We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach \u201cTracerSeq\u201d for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.", null, "pubmed:29700229", null, "CRISPR Targeted Embryo chordin A", "GSM3067201", null, "tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:chordin targeted Cas9", "CRISPR Targeted Embryo chordin A", "inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al. Nature Protocols 2017 and https://github.com/indrops/. Each sequencing spot is associated with a single biological read and 1 3 technical reads depending on the specific inDrops library preparation chemistry used. FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read. DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read. DEW101 208 used V3 chemistry in which read1 is the biological read read2 carries the first half of the cell barcode read3 carries the library index and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 \"Identify abundant barcodes\" see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1 parameters: n 1 l 15 e 200 m 200 best strata a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads. This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table. UMI counts were adjusted by a total counts normalization. For TracerSeq embryos inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above. These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt. An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv. A table of annotations for each ClusterID. Convert DEW to SRR csv. A table for converting between DEW and NCBI library names. CellTracerCounts csv. A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.", "Zebrafish Embryo Dissociated Cells", null, "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", null, "strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:chordin targeted Cas9", "GSM3067201", "GSM3067201: CRISPR Targeted Embryo chordin A; Danio rerio; RNA Seq", "GSM3067201", null, "1", "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", "GEO Accession:GSM3067201", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP136369", null, "loader:fastq load.py|options: appendBCtoName platform=Illumina", "DEW193.fastq.gz", "fastq", 1319530737.0, 22907290.0, "GSM3067201 r1", "0:57.60", "A:357858623;C:262292188;G:266165318;T:433212743;N:1865", 57, null, null, null, 357858623, 262292188, 266165318, 433212743, 1865, "SRX3841330", "SRS3087490", "SRA672434", "GEO", "Systems Biology, Harvard Medical School", 1, 0.8749, null, 0.13612, null, 0.79691, null, 0.51509, null, 61, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2018-03-23", "Segmentation", "Embryo", "Embryo Imprecise", "All anatomical structures"], [47772, "SRR6890900", "SRX3841330", "SRS3087490", "SRP136369", "PRJNA445487", "Systematic mapping of cell state trajectories cell lineage and perturbations in the zebrafish embryo using single cell transcriptomics", "GSE112294", "Transcriptome Analysis", "High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease. Here we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach we mapped a cell state landscape that describes axis patterning germ layer formation and organogenesis. We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach \u201cTracerSeq\u201d for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.", null, "pubmed:29700229", null, "CRISPR Targeted Embryo chordin A", "GSM3067201", null, "tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:chordin targeted Cas9", "CRISPR Targeted Embryo chordin A", "inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al. Nature Protocols 2017 and https://github.com/indrops/. Each sequencing spot is associated with a single biological read and 1 3 technical reads depending on the specific inDrops library preparation chemistry used. FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read. DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read. DEW101 208 used V3 chemistry in which read1 is the biological read read2 carries the first half of the cell barcode read3 carries the library index and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 \"Identify abundant barcodes\" see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1 parameters: n 1 l 15 e 200 m 200 best strata a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads. This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table. UMI counts were adjusted by a total counts normalization. For TracerSeq embryos inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above. These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt. An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv. A table of annotations for each ClusterID. Convert DEW to SRR csv. A table for converting between DEW and NCBI library names. CellTracerCounts csv. A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.", "Zebrafish Embryo Dissociated Cells", null, "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", null, "strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:chordin targeted Cas9", "GSM3067201", "GSM3067201: CRISPR Targeted Embryo chordin A; Danio rerio; RNA Seq", "GSM3067201", null, "1", "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", "GEO Accession:GSM3067201", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP136369", null, "loader:fastq load.py|options: appendBCtoName platform=Illumina", "DEW194.fastq.gz", "fastq", 1291827031.0, 22431130.0, "GSM3067201 r2", "0:57.59", "A:350651901;C:256281684;G:263900265;T:420991184;N:1997", 57, null, null, null, 350651901, 256281684, 263900265, 420991184, 1997, "SRX3841330", "SRS3087490", "SRA672434", "GEO", "Systems Biology, Harvard Medical School", 1, 0.87725, null, 0.13374, null, 0.79651, null, 0.53079, null, 41, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2018-03-23", "Segmentation", "Embryo", "Embryo Imprecise", "All anatomical structures"], [47773, "SRR6890901", "SRX3841330", "SRS3087490", "SRP136369", "PRJNA445487", "Systematic mapping of cell state trajectories cell lineage and perturbations in the zebrafish embryo using single cell transcriptomics", "GSE112294", "Transcriptome Analysis", "High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease. Here we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach we mapped a cell state landscape that describes axis patterning germ layer formation and organogenesis. We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach \u201cTracerSeq\u201d for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.", null, "pubmed:29700229", null, "CRISPR Targeted Embryo chordin A", "GSM3067201", null, "tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:chordin targeted Cas9", "CRISPR Targeted Embryo chordin A", "inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al. Nature Protocols 2017 and https://github.com/indrops/. Each sequencing spot is associated with a single biological read and 1 3 technical reads depending on the specific inDrops library preparation chemistry used. FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read. DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read. DEW101 208 used V3 chemistry in which read1 is the biological read read2 carries the first half of the cell barcode read3 carries the library index and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 \"Identify abundant barcodes\" see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1 parameters: n 1 l 15 e 200 m 200 best strata a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads. This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table. UMI counts were adjusted by a total counts normalization. For TracerSeq embryos inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above. These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt. An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv. A table of annotations for each ClusterID. Convert DEW to SRR csv. A table for converting between DEW and NCBI library names. CellTracerCounts csv. A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.", "Zebrafish Embryo Dissociated Cells", null, "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", null, "strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:chordin targeted Cas9", "GSM3067201", "GSM3067201: CRISPR Targeted Embryo chordin A; Danio rerio; RNA Seq", "GSM3067201", null, "1", "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", "GEO Accession:GSM3067201", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP136369", null, "loader:fastq load.py|options: appendBCtoName platform=Illumina", "DEW195.fastq.gz", "fastq", 1227344744.0, 21231899.0, "GSM3067201 r3", "0:57.81", "A:334536880;C:242949864;G:245894782;T:403961308;N:1910", 57, null, null, null, 334536880, 242949864, 245894782, 403961308, 1910, "SRX3841330", "SRS3087490", "SRA672434", "GEO", "Systems Biology, Harvard Medical School", 1, 0.87642, null, 0.13817, null, 0.79397, null, 0.52786, null, 61, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2018-03-23", "Segmentation", "Embryo", "Embryo Imprecise", "All anatomical structures"], [47774, "SRR6890902", "SRX3841330", "SRS3087490", "SRP136369", "PRJNA445487", "Systematic mapping of cell state trajectories cell lineage and perturbations in the zebrafish embryo using single cell transcriptomics", "GSE112294", "Transcriptome Analysis", "High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease. Here we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach we mapped a cell state landscape that describes axis patterning germ layer formation and organogenesis. We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach \u201cTracerSeq\u201d for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.", null, "pubmed:29700229", null, "CRISPR Targeted Embryo chordin A", "GSM3067201", null, "tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:chordin targeted Cas9", "CRISPR Targeted Embryo chordin A", "inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al. Nature Protocols 2017 and https://github.com/indrops/. Each sequencing spot is associated with a single biological read and 1 3 technical reads depending on the specific inDrops library preparation chemistry used. FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read. DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read. DEW101 208 used V3 chemistry in which read1 is the biological read read2 carries the first half of the cell barcode read3 carries the library index and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 \"Identify abundant barcodes\" see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1 parameters: n 1 l 15 e 200 m 200 best strata a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads. This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table. UMI counts were adjusted by a total counts normalization. For TracerSeq embryos inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above. These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt. An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv. A table of annotations for each ClusterID. Convert DEW to SRR csv. A table for converting between DEW and NCBI library names. CellTracerCounts csv. A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.", "Zebrafish Embryo Dissociated Cells", null, "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", null, "strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:chordin targeted Cas9", "GSM3067201", "GSM3067201: CRISPR Targeted Embryo chordin A; Danio rerio; RNA Seq", "GSM3067201", null, "1", "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", "GEO Accession:GSM3067201", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP136369", null, "loader:fastq load.py|options: appendBCtoName platform=Illumina", "DEW196.fastq.gz", "fastq", 1311849779.0, 22690047.0, "GSM3067201 r4", "0:57.82", "A:356392127;C:259146968;G:263404525;T:432904086;N:2073", 57, null, null, null, 356392127, 259146968, 263404525, 432904086, 2073, "SRX3841330", "SRS3087490", "SRA672434", "GEO", "Systems Biology, Harvard Medical School", 1, 0.88119, null, 0.13756, null, 0.79409, null, 0.52718, null, 61, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2018-03-23", "Segmentation", "Embryo", "Embryo Imprecise", "All anatomical structures"], [47775, "SRR6890903", "SRX3841330", "SRS3087490", "SRP136369", "PRJNA445487", "Systematic mapping of cell state trajectories cell lineage and perturbations in the zebrafish embryo using single cell transcriptomics", "GSE112294", "Transcriptome Analysis", "High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease. Here we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach we mapped a cell state landscape that describes axis patterning germ layer formation and organogenesis. We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach \u201cTracerSeq\u201d for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.", null, "pubmed:29700229", null, "CRISPR Targeted Embryo chordin A", "GSM3067201", null, "tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:chordin targeted Cas9", "CRISPR Targeted Embryo chordin A", "inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al. Nature Protocols 2017 and https://github.com/indrops/. Each sequencing spot is associated with a single biological read and 1 3 technical reads depending on the specific inDrops library preparation chemistry used. FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read. DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read. DEW101 208 used V3 chemistry in which read1 is the biological read read2 carries the first half of the cell barcode read3 carries the library index and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 \"Identify abundant barcodes\" see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1 parameters: n 1 l 15 e 200 m 200 best strata a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads. This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table. UMI counts were adjusted by a total counts normalization. For TracerSeq embryos inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above. These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt. An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv. A table of annotations for each ClusterID. Convert DEW to SRR csv. A table for converting between DEW and NCBI library names. CellTracerCounts csv. A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.", "Zebrafish Embryo Dissociated Cells", null, "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", null, "strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:chordin targeted Cas9", "GSM3067201", "GSM3067201: CRISPR Targeted Embryo chordin A; Danio rerio; RNA Seq", "GSM3067201", null, "1", "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", "GEO Accession:GSM3067201", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP136369", null, "loader:fastq load.py|options: appendBCtoName platform=Illumina", "DEW197.fastq.gz", "fastq", 1346161233.0, 23336788.0, "GSM3067201 r5", "0:57.68", "A:366046254;C:265311327;G:272129691;T:442671952;N:2009", 57, null, null, null, 366046254, 265311327, 272129691, 442671952, 2009, "SRX3841330", "SRS3087490", "SRA672434", "GEO", "Systems Biology, Harvard Medical School", 1, 0.87736, null, 0.13479, null, 0.79255, null, 0.53336, null, 60, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2018-03-23", "Segmentation", "Embryo", "Embryo Imprecise", "All anatomical structures"], [47776, "SRR6890896", "SRX3841329", "SRS3087489", "SRP136369", "PRJNA445487", "Systematic mapping of cell state trajectories cell lineage and perturbations in the zebrafish embryo using single cell transcriptomics", "GSE112294", "Transcriptome Analysis", "High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease. Here we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach we mapped a cell state landscape that describes axis patterning germ layer formation and organogenesis. We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach \u201cTracerSeq\u201d for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.", null, "pubmed:29700229", null, "TracerSeq Embryo 5", "GSM3067200", null, "tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type", "TracerSeq Embryo 5", "inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al. Nature Protocols 2017 and https://github.com/indrops/. Each sequencing spot is associated with a single biological read and 1 3 technical reads depending on the specific inDrops library preparation chemistry used. FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read. DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read. DEW101 208 used V3 chemistry in which read1 is the biological read read2 carries the first half of the cell barcode read3 carries the library index and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 \"Identify abundant barcodes\" see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1 parameters: n 1 l 15 e 200 m 200 best strata a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads. This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table. UMI counts were adjusted by a total counts normalization. For TracerSeq embryos inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above. These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt. An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv. A table of annotations for each ClusterID. Convert DEW to SRR csv. A table for converting between DEW and NCBI library names. CellTracerCounts csv. A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.", "Zebrafish Embryo Dissociated Cells", null, "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", null, "strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type", "GSM3067200", "GSM3067200: TracerSeq Embryo 5; Danio rerio; RNA Seq", "GSM3067200", null, "1", "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", "GEO Accession:GSM3067200", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP136369", null, "loader:fastq load.py|options: appendBCtoName platform=Illumina", "DEW167.fastq.gz", "fastq", 811220746.0, 14099347.0, "GSM3067200 r1", "0:57.54", "A:226775040;C:163877565;G:173170450;T:247395403;N:2288", 57, null, null, null, 226775040, 163877565, 173170450, 247395403, 2288, "SRX3841329", "SRS3087489", "SRA672434", "GEO", "Systems Biology, Harvard Medical School", 1, 0.88233, null, 0.12759, null, 0.79082, null, 0.51035, null, 61, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2018-03-23", "Pharyngula", "Embryo", "Embryo Imprecise", "All anatomical structures"], [47777, "SRR6890897", "SRX3841329", "SRS3087489", "SRP136369", "PRJNA445487", "Systematic mapping of cell state trajectories cell lineage and perturbations in the zebrafish embryo using single cell transcriptomics", "GSE112294", "Transcriptome Analysis", "High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease. Here we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach we mapped a cell state landscape that describes axis patterning germ layer formation and organogenesis. We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach \u201cTracerSeq\u201d for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.", null, "pubmed:29700229", null, "TracerSeq Embryo 5", "GSM3067200", null, "tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type", "TracerSeq Embryo 5", "inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al. Nature Protocols 2017 and https://github.com/indrops/. Each sequencing spot is associated with a single biological read and 1 3 technical reads depending on the specific inDrops library preparation chemistry used. FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read. DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read. DEW101 208 used V3 chemistry in which read1 is the biological read read2 carries the first half of the cell barcode read3 carries the library index and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 \"Identify abundant barcodes\" see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1 parameters: n 1 l 15 e 200 m 200 best strata a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads. This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table. UMI counts were adjusted by a total counts normalization. For TracerSeq embryos inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above. These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt. An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv. A table of annotations for each ClusterID. Convert DEW to SRR csv. A table for converting between DEW and NCBI library names. CellTracerCounts csv. A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.", "Zebrafish Embryo Dissociated Cells", null, "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", null, "strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type", "GSM3067200", "GSM3067200: TracerSeq Embryo 5; Danio rerio; RNA Seq", "GSM3067200", null, "1", "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", "GEO Accession:GSM3067200", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP136369", null, "loader:fastq load.py|options: appendBCtoName platform=Illumina", "DEW168.fastq.gz", "fastq", 824982674.0, 14331143.0, "GSM3067200 r2", "0:57.57", "A:231177547;C:167432521;G:176426897;T:249943460;N:2249", 57, null, null, null, 231177547, 167432521, 176426897, 249943460, 2249, "SRX3841329", "SRS3087489", "SRA672434", "GEO", "Systems Biology, Harvard Medical School", 1, 0.88583, null, 0.12411, null, 0.79277, null, 0.52031, null, 61, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2018-03-23", "Pharyngula", "Embryo", "Embryo Imprecise", "All anatomical structures"], [47778, "SRR6890898", "SRX3841329", "SRS3087489", "SRP136369", "PRJNA445487", "Systematic mapping of cell state trajectories cell lineage and perturbations in the zebrafish embryo using single cell transcriptomics", "GSE112294", "Transcriptome Analysis", "High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease. Here we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach we mapped a cell state landscape that describes axis patterning germ layer formation and organogenesis. We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach \u201cTracerSeq\u201d for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.", null, "pubmed:29700229", null, "TracerSeq Embryo 5", "GSM3067200", null, "tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type", "TracerSeq Embryo 5", "inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al. Nature Protocols 2017 and https://github.com/indrops/. Each sequencing spot is associated with a single biological read and 1 3 technical reads depending on the specific inDrops library preparation chemistry used. FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read. DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read. DEW101 208 used V3 chemistry in which read1 is the biological read read2 carries the first half of the cell barcode read3 carries the library index and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 \"Identify abundant barcodes\" see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1 parameters: n 1 l 15 e 200 m 200 best strata a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads. This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table. UMI counts were adjusted by a total counts normalization. For TracerSeq embryos inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above. These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt. An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv. A table of annotations for each ClusterID. Convert DEW to SRR csv. A table for converting between DEW and NCBI library names. CellTracerCounts csv. A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.", "Zebrafish Embryo Dissociated Cells", null, "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", null, "strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type", "GSM3067200", "GSM3067200: TracerSeq Embryo 5; Danio rerio; RNA Seq", "GSM3067200", null, "1", "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", "GEO Accession:GSM3067200", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP136369", null, "loader:fastq load.py|options: appendBCtoName platform=Illumina", "DEW169.fastq.gz", "fastq", 693186309.0, 12065222.0, "GSM3067200 r3", "0:57.45", "A:197516210;C:138205483;G:144601216;T:212861628;N:1772", 57, null, null, null, 197516210, 138205483, 144601216, 212861628, 1772, "SRX3841329", "SRS3087489", "SRA672434", "GEO", "Systems Biology, Harvard Medical School", 1, 0.86621, null, 0.12722, null, 0.78823, null, 0.51859, null, 61, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2018-03-23", "Pharyngula", "Embryo", "Embryo Imprecise", "All anatomical structures"], [47779, "SRR11699740", "SRX3841329", "SRS3087489", "SRP136369", "PRJNA445487", "Systematic mapping of cell state trajectories cell lineage and perturbations in the zebrafish embryo using single cell transcriptomics", "GSE112294", "Transcriptome Analysis", "High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease. Here we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach we mapped a cell state landscape that describes axis patterning germ layer formation and organogenesis. We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach \u201cTracerSeq\u201d for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.", null, "pubmed:29700229", null, "TracerSeq Embryo 5", "GSM3067200", null, "tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type", "TracerSeq Embryo 5", "inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al. Nature Protocols 2017 and https://github.com/indrops/. Each sequencing spot is associated with a single biological read and 1 3 technical reads depending on the specific inDrops library preparation chemistry used. FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read. DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read. DEW101 208 used V3 chemistry in which read1 is the biological read read2 carries the first half of the cell barcode read3 carries the library index and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 \"Identify abundant barcodes\" see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1 parameters: n 1 l 15 e 200 m 200 best strata a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads. This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table. UMI counts were adjusted by a total counts normalization. For TracerSeq embryos inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above. These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt. An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv. A table of annotations for each ClusterID. Convert DEW to SRR csv. A table for converting between DEW and NCBI library names. CellTracerCounts csv. A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.", "Zebrafish Embryo Dissociated Cells", null, "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", null, "strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type", "GSM3067200", "GSM3067200: TracerSeq Embryo 5; Danio rerio; RNA Seq", "GSM3067200", null, "1", "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", "GEO Accession:GSM3067200", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP136369", null, "loader:fastq load.py|options: appendBCtoName", "DEW167.gfp.fastq.gz", "fastq", 637401493.0, 11247658.0, "GSM3067200 r11", "0:56.67", "A:211763567;C:130934906;G:157778746;T:136920350;N:3924", 56, null, null, null, 211763567, 130934906, 157778746, 136920350, 3924, "SRX3841329", "SRS3087489", "SRA672434", "GEO", "Systems Biology, Harvard Medical School", 1, 0.00818, null, 0.0001, null, 0.99902, null, 0.79816, null, 61, null, "T", null, "under 1.2% mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2018-03-23", "Pharyngula", "Embryo", "Embryo Imprecise", "All anatomical structures"], [47780, "SRR11699741", "SRX3841329", "SRS3087489", "SRP136369", "PRJNA445487", "Systematic mapping of cell state trajectories cell lineage and perturbations in the zebrafish embryo using single cell transcriptomics", "GSE112294", "Transcriptome Analysis", "High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease. Here we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach we mapped a cell state landscape that describes axis patterning germ layer formation and organogenesis. We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach \u201cTracerSeq\u201d for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.", null, "pubmed:29700229", null, "TracerSeq Embryo 5", "GSM3067200", null, "tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type", "TracerSeq Embryo 5", "inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al. Nature Protocols 2017 and https://github.com/indrops/. Each sequencing spot is associated with a single biological read and 1 3 technical reads depending on the specific inDrops library preparation chemistry used. FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read. DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read. DEW101 208 used V3 chemistry in which read1 is the biological read read2 carries the first half of the cell barcode read3 carries the library index and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 \"Identify abundant barcodes\" see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1 parameters: n 1 l 15 e 200 m 200 best strata a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads. This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table. UMI counts were adjusted by a total counts normalization. For TracerSeq embryos inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above. These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt. An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv. A table of annotations for each ClusterID. Convert DEW to SRR csv. A table for converting between DEW and NCBI library names. CellTracerCounts csv. A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.", "Zebrafish Embryo Dissociated Cells", null, "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", null, "strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type", "GSM3067200", "GSM3067200: TracerSeq Embryo 5; Danio rerio; RNA Seq", "GSM3067200", null, "1", "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", "GEO Accession:GSM3067200", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP136369", null, "loader:fastq load.py|options: appendBCtoName", "DEW168.gfp.fastq.gz", "fastq", 648934432.0, 11464121.0, "GSM3067200 r12", "0:56.61", "A:211915696;C:134836103;G:161336635;T:140841785;N:4213", 56, null, null, null, 211915696, 134836103, 161336635, 140841785, 4213, "SRX3841329", "SRS3087489", "SRA672434", "GEO", "Systems Biology, Harvard Medical School", 1, 0.0103, null, 0.00018, null, 0.99853, null, 0.78199, null, 61, null, "T", null, "under 1.2% mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2018-03-23", "Pharyngula", "Embryo", "Embryo Imprecise", "All anatomical structures"], [47781, "SRR11699742", "SRX3841329", "SRS3087489", "SRP136369", "PRJNA445487", "Systematic mapping of cell state trajectories cell lineage and perturbations in the zebrafish embryo using single cell transcriptomics", "GSE112294", "Transcriptome Analysis", "High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease. Here we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach we mapped a cell state landscape that describes axis patterning germ layer formation and organogenesis. We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach \u201cTracerSeq\u201d for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.", null, "pubmed:29700229", null, "TracerSeq Embryo 5", "GSM3067200", null, "tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type", "TracerSeq Embryo 5", "inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al. Nature Protocols 2017 and https://github.com/indrops/. Each sequencing spot is associated with a single biological read and 1 3 technical reads depending on the specific inDrops library preparation chemistry used. FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read. DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read. DEW101 208 used V3 chemistry in which read1 is the biological read read2 carries the first half of the cell barcode read3 carries the library index and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 \"Identify abundant barcodes\" see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1 parameters: n 1 l 15 e 200 m 200 best strata a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads. This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table. UMI counts were adjusted by a total counts normalization. For TracerSeq embryos inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above. These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt. An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv. A table of annotations for each ClusterID. Convert DEW to SRR csv. A table for converting between DEW and NCBI library names. CellTracerCounts csv. A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.", "Zebrafish Embryo Dissociated Cells", null, "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", null, "strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type", "GSM3067200", "GSM3067200: TracerSeq Embryo 5; Danio rerio; RNA Seq", "GSM3067200", null, "1", "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", "GEO Accession:GSM3067200", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP136369", null, "loader:fastq load.py|options: appendBCtoName", "DEW169.gfp.fastq.gz", "fastq", 555982541.0, 9807444.0, "GSM3067200 r13", "0:56.69", "A:182949392;C:115632932;G:137856329;T:119540249;N:3639", 56, null, null, null, 182949392, 115632932, 137856329, 119540249, 3639, "SRX3841329", "SRS3087489", "SRA672434", "GEO", "Systems Biology, Harvard Medical School", 1, 0.01235, null, 0.00032, null, 0.99868, null, 0.81431, null, 61, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2018-03-23", "Pharyngula", "Embryo", "Embryo Imprecise", "All anatomical structures"], [47782, "SRR6890891", "SRX3841328", "SRS3087488", "SRP136369", "PRJNA445487", "Systematic mapping of cell state trajectories cell lineage and perturbations in the zebrafish embryo using single cell transcriptomics", "GSE112294", "Transcriptome Analysis", "High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease. Here we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach we mapped a cell state landscape that describes axis patterning germ layer formation and organogenesis. We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach \u201cTracerSeq\u201d for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.", null, "pubmed:29700229", null, "TracerSeq Embryo 4", "GSM3067199", null, "tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type", "TracerSeq Embryo 4", "inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al. Nature Protocols 2017 and https://github.com/indrops/. Each sequencing spot is associated with a single biological read and 1 3 technical reads depending on the specific inDrops library preparation chemistry used. FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read. DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read. DEW101 208 used V3 chemistry in which read1 is the biological read read2 carries the first half of the cell barcode read3 carries the library index and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 \"Identify abundant barcodes\" see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1 parameters: n 1 l 15 e 200 m 200 best strata a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads. This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table. UMI counts were adjusted by a total counts normalization. For TracerSeq embryos inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above. These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt. An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv. A table of annotations for each ClusterID. Convert DEW to SRR csv. A table for converting between DEW and NCBI library names. CellTracerCounts csv. A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.", "Zebrafish Embryo Dissociated Cells", null, "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", null, "strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type", "GSM3067199", "GSM3067199: TracerSeq Embryo 4; Danio rerio; RNA Seq", "GSM3067199", null, "1", "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", "GEO Accession:GSM3067199", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP136369", null, "loader:fastq load.py|options: appendBCtoName platform=Illumina", "DEW162.fastq.gz", "fastq", 884280521.0, 15352773.0, "GSM3067199 r1", "0:57.60", "A:248995548;C:176713956;G:184425201;T:274143441;N:2375", 57, null, null, null, 248995548, 176713956, 184425201, 274143441, 2375, "SRX3841328", "SRS3087488", "SRA672434", "GEO", "Systems Biology, Harvard Medical School", 1, 0.87729, null, 0.1278, null, 0.77904, null, 0.51481, null, 61, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2018-03-23", "Pharyngula", "Embryo", "Embryo Imprecise", "All anatomical structures"], [47783, "SRR6890892", "SRX3841328", "SRS3087488", "SRP136369", "PRJNA445487", "Systematic mapping of cell state trajectories cell lineage and perturbations in the zebrafish embryo using single cell transcriptomics", "GSE112294", "Transcriptome Analysis", "High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease. Here we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach we mapped a cell state landscape that describes axis patterning germ layer formation and organogenesis. We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach \u201cTracerSeq\u201d for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.", null, "pubmed:29700229", null, "TracerSeq Embryo 4", "GSM3067199", null, "tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type", "TracerSeq Embryo 4", "inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al. Nature Protocols 2017 and https://github.com/indrops/. Each sequencing spot is associated with a single biological read and 1 3 technical reads depending on the specific inDrops library preparation chemistry used. FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read. DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read. DEW101 208 used V3 chemistry in which read1 is the biological read read2 carries the first half of the cell barcode read3 carries the library index and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 \"Identify abundant barcodes\" see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1 parameters: n 1 l 15 e 200 m 200 best strata a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads. This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table. UMI counts were adjusted by a total counts normalization. For TracerSeq embryos inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above. These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt. An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv. A table of annotations for each ClusterID. Convert DEW to SRR csv. A table for converting between DEW and NCBI library names. CellTracerCounts csv. A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.", "Zebrafish Embryo Dissociated Cells", null, "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", null, "strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type", "GSM3067199", "GSM3067199: TracerSeq Embryo 4; Danio rerio; RNA Seq", "GSM3067199", null, "1", "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", "GEO Accession:GSM3067199", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP136369", null, "loader:fastq load.py|options: appendBCtoName platform=Illumina", "DEW163.fastq.gz", "fastq", 952171001.0, 16579450.0, "GSM3067199 r2", "0:57.43", "A:269068875;C:192967485;G:199258236;T:290873928;N:2477", 57, null, null, null, 269068875, 192967485, 199258236, 290873928, 2477, "SRX3841328", "SRS3087488", "SRA672434", "GEO", "Systems Biology, Harvard Medical School", 1, 0.86963, null, 0.12299, null, 0.78261, null, 0.51621, null, 61, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2018-03-23", "Pharyngula", "Embryo", "Embryo Imprecise", "All anatomical structures"], [47784, "SRR6890893", "SRX3841328", "SRS3087488", "SRP136369", "PRJNA445487", "Systematic mapping of cell state trajectories cell lineage and perturbations in the zebrafish embryo using single cell transcriptomics", "GSE112294", "Transcriptome Analysis", "High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease. Here we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach we mapped a cell state landscape that describes axis patterning germ layer formation and organogenesis. We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach \u201cTracerSeq\u201d for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.", null, "pubmed:29700229", null, "TracerSeq Embryo 4", "GSM3067199", null, "tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type", "TracerSeq Embryo 4", "inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al. Nature Protocols 2017 and https://github.com/indrops/. Each sequencing spot is associated with a single biological read and 1 3 technical reads depending on the specific inDrops library preparation chemistry used. FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read. DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read. DEW101 208 used V3 chemistry in which read1 is the biological read read2 carries the first half of the cell barcode read3 carries the library index and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 \"Identify abundant barcodes\" see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1 parameters: n 1 l 15 e 200 m 200 best strata a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads. This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table. UMI counts were adjusted by a total counts normalization. For TracerSeq embryos inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above. These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt. An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv. A table of annotations for each ClusterID. Convert DEW to SRR csv. A table for converting between DEW and NCBI library names. CellTracerCounts csv. A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.", "Zebrafish Embryo Dissociated Cells", null, "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", null, "strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type", "GSM3067199", "GSM3067199: TracerSeq Embryo 4; Danio rerio; RNA Seq", "GSM3067199", null, "1", "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", "GEO Accession:GSM3067199", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP136369", null, "loader:fastq load.py|options: appendBCtoName platform=Illumina", "DEW164.fastq.gz", "fastq", 1133026695.0, 19698928.0, "GSM3067199 r3", "0:57.52", "A:320544827;C:227286162;G:235085716;T:350106942;N:3048", 57, null, null, null, 320544827, 227286162, 235085716, 350106942, 3048, "SRX3841328", "SRS3087488", "SRA672434", "GEO", "Systems Biology, Harvard Medical School", 1, 0.87678, null, 0.12527, null, 0.78169, null, 0.50623, null, 61, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2018-03-23", "Pharyngula", "Embryo", "Embryo Imprecise", "All anatomical structures"], [47785, "SRR6890894", "SRX3841328", "SRS3087488", "SRP136369", "PRJNA445487", "Systematic mapping of cell state trajectories cell lineage and perturbations in the zebrafish embryo using single cell transcriptomics", "GSE112294", "Transcriptome Analysis", "High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease. Here we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach we mapped a cell state landscape that describes axis patterning germ layer formation and organogenesis. We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach \u201cTracerSeq\u201d for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.", null, "pubmed:29700229", null, "TracerSeq Embryo 4", "GSM3067199", null, "tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type", "TracerSeq Embryo 4", "inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al. Nature Protocols 2017 and https://github.com/indrops/. Each sequencing spot is associated with a single biological read and 1 3 technical reads depending on the specific inDrops library preparation chemistry used. FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read. DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read. DEW101 208 used V3 chemistry in which read1 is the biological read read2 carries the first half of the cell barcode read3 carries the library index and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 \"Identify abundant barcodes\" see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1 parameters: n 1 l 15 e 200 m 200 best strata a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads. This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table. UMI counts were adjusted by a total counts normalization. For TracerSeq embryos inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above. These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt. An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv. A table of annotations for each ClusterID. Convert DEW to SRR csv. A table for converting between DEW and NCBI library names. CellTracerCounts csv. A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.", "Zebrafish Embryo Dissociated Cells", null, "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", null, "strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type", "GSM3067199", "GSM3067199: TracerSeq Embryo 4; Danio rerio; RNA Seq", "GSM3067199", null, "1", "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", "GEO Accession:GSM3067199", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP136369", null, "loader:fastq load.py|options: appendBCtoName platform=Illumina", "DEW165.fastq.gz", "fastq", 793581204.0, 13818281.0, "GSM3067199 r4", "0:57.43", "A:223975396;C:158365631;G:165125978;T:246112109;N:2090", 57, null, null, null, 223975396, 158365631, 165125978, 246112109, 2090, "SRX3841328", "SRS3087488", "SRA672434", "GEO", "Systems Biology, Harvard Medical School", 1, 0.87803, null, 0.12874, null, 0.78374, null, 0.50983, null, 44, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2018-03-23", "Pharyngula", "Embryo", "Embryo Imprecise", "All anatomical structures"], [47786, "SRR6890895", "SRX3841328", "SRS3087488", "SRP136369", "PRJNA445487", "Systematic mapping of cell state trajectories cell lineage and perturbations in the zebrafish embryo using single cell transcriptomics", "GSE112294", "Transcriptome Analysis", "High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease. Here we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach we mapped a cell state landscape that describes axis patterning germ layer formation and organogenesis. We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach \u201cTracerSeq\u201d for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.", null, "pubmed:29700229", null, "TracerSeq Embryo 4", "GSM3067199", null, "tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type", "TracerSeq Embryo 4", "inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al. Nature Protocols 2017 and https://github.com/indrops/. Each sequencing spot is associated with a single biological read and 1 3 technical reads depending on the specific inDrops library preparation chemistry used. FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read. DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read. DEW101 208 used V3 chemistry in which read1 is the biological read read2 carries the first half of the cell barcode read3 carries the library index and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 \"Identify abundant barcodes\" see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1 parameters: n 1 l 15 e 200 m 200 best strata a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads. This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table. UMI counts were adjusted by a total counts normalization. For TracerSeq embryos inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above. These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt. An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv. A table of annotations for each ClusterID. Convert DEW to SRR csv. A table for converting between DEW and NCBI library names. CellTracerCounts csv. A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.", "Zebrafish Embryo Dissociated Cells", null, "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", null, "strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type", "GSM3067199", "GSM3067199: TracerSeq Embryo 4; Danio rerio; RNA Seq", "GSM3067199", null, "1", "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", "GEO Accession:GSM3067199", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP136369", null, "loader:fastq load.py|options: appendBCtoName platform=Illumina", "DEW166.fastq.gz", "fastq", 792728001.0, 13800321.0, "GSM3067199 r5", "0:57.44", "A:225238995;C:161675115;G:167314732;T:238497072;N:2087", 57, null, null, null, 225238995, 161675115, 167314732, 238497072, 2087, "SRX3841328", "SRS3087488", "SRA672434", "GEO", "Systems Biology, Harvard Medical School", 1, 0.87776, null, 0.11531, null, 0.79458, null, 0.53005, null, 61, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2018-03-23", "Pharyngula", "Embryo", "Embryo Imprecise", "All anatomical structures"], [47787, "SRR11699735", "SRX3841328", "SRS3087488", "SRP136369", "PRJNA445487", "Systematic mapping of cell state trajectories cell lineage and perturbations in the zebrafish embryo using single cell transcriptomics", "GSE112294", "Transcriptome Analysis", "High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease. Here we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach we mapped a cell state landscape that describes axis patterning germ layer formation and organogenesis. We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach \u201cTracerSeq\u201d for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.", null, "pubmed:29700229", null, "TracerSeq Embryo 4", "GSM3067199", null, "tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type", "TracerSeq Embryo 4", "inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al. Nature Protocols 2017 and https://github.com/indrops/. Each sequencing spot is associated with a single biological read and 1 3 technical reads depending on the specific inDrops library preparation chemistry used. FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read. DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read. DEW101 208 used V3 chemistry in which read1 is the biological read read2 carries the first half of the cell barcode read3 carries the library index and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 \"Identify abundant barcodes\" see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1 parameters: n 1 l 15 e 200 m 200 best strata a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads. This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table. UMI counts were adjusted by a total counts normalization. For TracerSeq embryos inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above. These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt. An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv. A table of annotations for each ClusterID. Convert DEW to SRR csv. A table for converting between DEW and NCBI library names. CellTracerCounts csv. A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.", "Zebrafish Embryo Dissociated Cells", null, "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", null, "strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type", "GSM3067199", "GSM3067199: TracerSeq Embryo 4; Danio rerio; RNA Seq", "GSM3067199", null, "1", "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", "GEO Accession:GSM3067199", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP136369", null, "loader:fastq load.py|options: appendBCtoName", "DEW162.gfp.fastq.gz", "fastq", 725258705.0, 12804354.0, "GSM3067199 r11", "0:56.64", "A:244821707;C:144850405;G:178149916;T:157432024;N:4653", 56, null, null, null, 244821707, 144850405, 178149916, 157432024, 4653, "SRX3841328", "SRS3087488", "SRA672434", "GEO", "Systems Biology, Harvard Medical School", 1, 0.00953, null, 0.00045, null, 0.99874, null, 0.84025, null, 61, null, "T", null, "under 1.2% mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2018-03-23", "Pharyngula", "Embryo", "Embryo Imprecise", "All anatomical structures"], [47788, "SRR11699736", "SRX3841328", "SRS3087488", "SRP136369", "PRJNA445487", "Systematic mapping of cell state trajectories cell lineage and perturbations in the zebrafish embryo using single cell transcriptomics", "GSE112294", "Transcriptome Analysis", "High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease. Here we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach we mapped a cell state landscape that describes axis patterning germ layer formation and organogenesis. We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach \u201cTracerSeq\u201d for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.", null, "pubmed:29700229", null, "TracerSeq Embryo 4", "GSM3067199", null, "tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type", "TracerSeq Embryo 4", "inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al. Nature Protocols 2017 and https://github.com/indrops/. Each sequencing spot is associated with a single biological read and 1 3 technical reads depending on the specific inDrops library preparation chemistry used. FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read. DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read. DEW101 208 used V3 chemistry in which read1 is the biological read read2 carries the first half of the cell barcode read3 carries the library index and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 \"Identify abundant barcodes\" see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1 parameters: n 1 l 15 e 200 m 200 best strata a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads. This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table. UMI counts were adjusted by a total counts normalization. For TracerSeq embryos inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above. These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt. An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv. A table of annotations for each ClusterID. Convert DEW to SRR csv. A table for converting between DEW and NCBI library names. CellTracerCounts csv. A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.", "Zebrafish Embryo Dissociated Cells", null, "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", null, "strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type", "GSM3067199", "GSM3067199: TracerSeq Embryo 4; Danio rerio; RNA Seq", "GSM3067199", null, "1", "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", "GEO Accession:GSM3067199", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP136369", null, "loader:fastq load.py|options: appendBCtoName", "DEW163.gfp.fastq.gz", "fastq", 709146800.0, 12584798.0, "GSM3067199 r12", "0:56.35", "A:239870182;C:143213119;G:172165989;T:153892922;N:4588", 56, null, null, null, 239870182, 143213119, 172165989, 153892922, 4588, "SRX3841328", "SRS3087488", "SRA672434", "GEO", "Systems Biology, Harvard Medical School", 1, 0.00541, null, 0.00022, null, 0.99888, null, 0.84231, null, 61, null, "T", null, "under 1.2% mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2018-03-23", "Pharyngula", "Embryo", "Embryo Imprecise", "All anatomical structures"], [47789, "SRR11699737", "SRX3841328", "SRS3087488", "SRP136369", "PRJNA445487", "Systematic mapping of cell state trajectories cell lineage and perturbations in the zebrafish embryo using single cell transcriptomics", "GSE112294", "Transcriptome Analysis", "High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease. Here we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach we mapped a cell state landscape that describes axis patterning germ layer formation and organogenesis. We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach \u201cTracerSeq\u201d for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.", null, "pubmed:29700229", null, "TracerSeq Embryo 4", "GSM3067199", null, "tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type", "TracerSeq Embryo 4", "inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al. Nature Protocols 2017 and https://github.com/indrops/. Each sequencing spot is associated with a single biological read and 1 3 technical reads depending on the specific inDrops library preparation chemistry used. FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read. DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read. DEW101 208 used V3 chemistry in which read1 is the biological read read2 carries the first half of the cell barcode read3 carries the library index and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 \"Identify abundant barcodes\" see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1 parameters: n 1 l 15 e 200 m 200 best strata a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads. This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table. UMI counts were adjusted by a total counts normalization. For TracerSeq embryos inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above. These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt. An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv. A table of annotations for each ClusterID. Convert DEW to SRR csv. A table for converting between DEW and NCBI library names. CellTracerCounts csv. A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.", "Zebrafish Embryo Dissociated Cells", null, "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", null, "strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type", "GSM3067199", "GSM3067199: TracerSeq Embryo 4; Danio rerio; RNA Seq", "GSM3067199", null, "1", "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", "GEO Accession:GSM3067199", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP136369", null, "loader:fastq load.py|options: appendBCtoName", "DEW164.gfp.fastq.gz", "fastq", 648001757.0, 11476883.0, "GSM3067199 r13", "0:56.46", "A:220229172;C:129764679;G:155857424;T:142146491;N:3991", 56, null, null, null, 220229172, 129764679, 155857424, 142146491, 3991, "SRX3841328", "SRS3087488", "SRA672434", "GEO", "Systems Biology, Harvard Medical School", 1, 0.00638, null, 0.00028, null, 0.999, null, 0.82363, null, 53, null, "T", null, "under 1.2% mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2018-03-23", "Pharyngula", "Embryo", "Embryo Imprecise", "All anatomical structures"], [47790, "SRR11699738", "SRX3841328", "SRS3087488", "SRP136369", "PRJNA445487", "Systematic mapping of cell state trajectories cell lineage and perturbations in the zebrafish embryo using single cell transcriptomics", "GSE112294", "Transcriptome Analysis", "High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease. Here we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach we mapped a cell state landscape that describes axis patterning germ layer formation and organogenesis. We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach \u201cTracerSeq\u201d for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.", null, "pubmed:29700229", null, "TracerSeq Embryo 4", "GSM3067199", null, "tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type", "TracerSeq Embryo 4", "inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al. Nature Protocols 2017 and https://github.com/indrops/. Each sequencing spot is associated with a single biological read and 1 3 technical reads depending on the specific inDrops library preparation chemistry used. FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read. DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read. DEW101 208 used V3 chemistry in which read1 is the biological read read2 carries the first half of the cell barcode read3 carries the library index and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 \"Identify abundant barcodes\" see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1 parameters: n 1 l 15 e 200 m 200 best strata a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads. This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table. UMI counts were adjusted by a total counts normalization. For TracerSeq embryos inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above. These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt. An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv. A table of annotations for each ClusterID. Convert DEW to SRR csv. A table for converting between DEW and NCBI library names. CellTracerCounts csv. A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.", "Zebrafish Embryo Dissociated Cells", null, "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", null, "strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type", "GSM3067199", "GSM3067199: TracerSeq Embryo 4; Danio rerio; RNA Seq", "GSM3067199", null, "1", "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", "GEO Accession:GSM3067199", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP136369", null, "loader:fastq load.py|options: appendBCtoName", "DEW165.gfp.fastq.gz", "fastq", 596792506.0, 10531209.0, "GSM3067199 r14", "0:56.67", "A:200618246;C:122094142;G:144891967;T:129184490;N:3661", 56, null, null, null, 200618246, 122094142, 144891967, 129184490, 3661, "SRX3841328", "SRS3087488", "SRA672434", "GEO", "Systems Biology, Harvard Medical School", 1, 0.00789, null, 0.00046, null, 0.99888, null, 0.84071, null, 61, null, "T", null, "under 1.2% mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2018-03-23", "Pharyngula", "Embryo", "Embryo Imprecise", "All anatomical structures"], [47791, "SRR11699739", "SRX3841328", "SRS3087488", "SRP136369", "PRJNA445487", "Systematic mapping of cell state trajectories cell lineage and perturbations in the zebrafish embryo using single cell transcriptomics", "GSE112294", "Transcriptome Analysis", "High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease. Here we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach we mapped a cell state landscape that describes axis patterning germ layer formation and organogenesis. We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach \u201cTracerSeq\u201d for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.", null, "pubmed:29700229", null, "TracerSeq Embryo 4", "GSM3067199", null, "tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type", "TracerSeq Embryo 4", "inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al. Nature Protocols 2017 and https://github.com/indrops/. Each sequencing spot is associated with a single biological read and 1 3 technical reads depending on the specific inDrops library preparation chemistry used. FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read. DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read. DEW101 208 used V3 chemistry in which read1 is the biological read read2 carries the first half of the cell barcode read3 carries the library index and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 \"Identify abundant barcodes\" see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1 parameters: n 1 l 15 e 200 m 200 best strata a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads. This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table. UMI counts were adjusted by a total counts normalization. For TracerSeq embryos inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above. These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt. An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv. A table of annotations for each ClusterID. Convert DEW to SRR csv. A table for converting between DEW and NCBI library names. CellTracerCounts csv. A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.", "Zebrafish Embryo Dissociated Cells", null, "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", null, "strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type", "GSM3067199", "GSM3067199: TracerSeq Embryo 4; Danio rerio; RNA Seq", "GSM3067199", null, "1", "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", "GEO Accession:GSM3067199", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP136369", null, "loader:fastq load.py|options: appendBCtoName", "DEW166.gfp.fastq.gz", "fastq", 566854985.0, 10065909.0, "GSM3067199 r15", "0:56.31", "A:192383179;C:111486159;G:139172733;T:123809364;N:3550", 56, null, null, null, 192383179, 111486159, 139172733, 123809364, 3550, "SRX3841328", "SRS3087488", "SRA672434", "GEO", "Systems Biology, Harvard Medical School", 1, 0.00671, null, 0.00038, null, 0.99886, null, 0.85738, null, 61, null, "T", null, "under 1.2% mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2018-03-23", "Pharyngula", "Embryo", "Embryo Imprecise", "All anatomical structures"], [47792, "SRR6890887", "SRX3841327", "SRS3087487", "SRP136369", "PRJNA445487", "Systematic mapping of cell state trajectories cell lineage and perturbations in the zebrafish embryo using single cell transcriptomics", "GSE112294", "Transcriptome Analysis", "High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease. Here we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach we mapped a cell state landscape that describes axis patterning germ layer formation and organogenesis. We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach \u201cTracerSeq\u201d for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.", null, "pubmed:29700229", null, "TracerSeq Embryo 3", "GSM3067198", null, "tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type", "TracerSeq Embryo 3", "inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al. Nature Protocols 2017 and https://github.com/indrops/. Each sequencing spot is associated with a single biological read and 1 3 technical reads depending on the specific inDrops library preparation chemistry used. FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read. DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read. DEW101 208 used V3 chemistry in which read1 is the biological read read2 carries the first half of the cell barcode read3 carries the library index and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 \"Identify abundant barcodes\" see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1 parameters: n 1 l 15 e 200 m 200 best strata a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads. This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table. UMI counts were adjusted by a total counts normalization. For TracerSeq embryos inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above. These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt. An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv. A table of annotations for each ClusterID. Convert DEW to SRR csv. A table for converting between DEW and NCBI library names. CellTracerCounts csv. A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.", "Zebrafish Embryo Dissociated Cells", null, "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", null, "strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type", "GSM3067198", "GSM3067198: TracerSeq Embryo 3; Danio rerio; RNA Seq", "GSM3067198", null, "1", "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", "GEO Accession:GSM3067198", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP136369", null, "loader:fastq load.py|options: appendBCtoName platform=Illumina", "DEW158.fastq.gz", "fastq", 965976032.0, 16779564.0, "GSM3067198 r1", "0:57.57", "A:270793370;C:196343924;G:207973938;T:290862136;N:2664", 57, null, null, null, 270793370, 196343924, 207973938, 290862136, 2664, "SRX3841327", "SRS3087487", "SRA672434", "GEO", "Systems Biology, Harvard Medical School", 1, 0.88031, null, 0.11243, null, 0.7919, null, 0.51366, null, 61, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2018-03-23", "Pharyngula", "Embryo", "Embryo Imprecise", "All anatomical structures"], [47793, "SRR6890888", "SRX3841327", "SRS3087487", "SRP136369", "PRJNA445487", "Systematic mapping of cell state trajectories cell lineage and perturbations in the zebrafish embryo using single cell transcriptomics", "GSE112294", "Transcriptome Analysis", "High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease. Here we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach we mapped a cell state landscape that describes axis patterning germ layer formation and organogenesis. We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach \u201cTracerSeq\u201d for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.", null, "pubmed:29700229", null, "TracerSeq Embryo 3", "GSM3067198", null, "tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type", "TracerSeq Embryo 3", "inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al. Nature Protocols 2017 and https://github.com/indrops/. Each sequencing spot is associated with a single biological read and 1 3 technical reads depending on the specific inDrops library preparation chemistry used. FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read. DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read. DEW101 208 used V3 chemistry in which read1 is the biological read read2 carries the first half of the cell barcode read3 carries the library index and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 \"Identify abundant barcodes\" see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1 parameters: n 1 l 15 e 200 m 200 best strata a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads. This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table. UMI counts were adjusted by a total counts normalization. For TracerSeq embryos inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above. These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt. An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv. A table of annotations for each ClusterID. Convert DEW to SRR csv. A table for converting between DEW and NCBI library names. CellTracerCounts csv. A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.", "Zebrafish Embryo Dissociated Cells", null, "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", null, "strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type", "GSM3067198", "GSM3067198: TracerSeq Embryo 3; Danio rerio; RNA Seq", "GSM3067198", null, "1", "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", "GEO Accession:GSM3067198", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP136369", null, "loader:fastq load.py|options: appendBCtoName platform=Illumina", "DEW159.fastq.gz", "fastq", 1015306225.0, 17632016.0, "GSM3067198 r2", "0:57.58", "A:282554762;C:206663548;G:220061729;T:306023342;N:2844", 57, null, null, null, 282554762, 206663548, 220061729, 306023342, 2844, "SRX3841327", "SRS3087487", "SRA672434", "GEO", "Systems Biology, Harvard Medical School", 1, 0.88781, null, 0.11491, null, 0.79251, null, 0.51135, null, 61, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2018-03-23", "Pharyngula", "Embryo", "Embryo Imprecise", "All anatomical structures"], [47794, "SRR6890889", "SRX3841327", "SRS3087487", "SRP136369", "PRJNA445487", "Systematic mapping of cell state trajectories cell lineage and perturbations in the zebrafish embryo using single cell transcriptomics", "GSE112294", "Transcriptome Analysis", "High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease. Here we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach we mapped a cell state landscape that describes axis patterning germ layer formation and organogenesis. We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach \u201cTracerSeq\u201d for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.", null, "pubmed:29700229", null, "TracerSeq Embryo 3", "GSM3067198", null, "tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type", "TracerSeq Embryo 3", "inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al. Nature Protocols 2017 and https://github.com/indrops/. Each sequencing spot is associated with a single biological read and 1 3 technical reads depending on the specific inDrops library preparation chemistry used. FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read. DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read. DEW101 208 used V3 chemistry in which read1 is the biological read read2 carries the first half of the cell barcode read3 carries the library index and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 \"Identify abundant barcodes\" see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1 parameters: n 1 l 15 e 200 m 200 best strata a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads. This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table. UMI counts were adjusted by a total counts normalization. For TracerSeq embryos inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above. These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt. An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv. A table of annotations for each ClusterID. Convert DEW to SRR csv. A table for converting between DEW and NCBI library names. CellTracerCounts csv. A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.", "Zebrafish Embryo Dissociated Cells", null, "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", null, "strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type", "GSM3067198", "GSM3067198: TracerSeq Embryo 3; Danio rerio; RNA Seq", "GSM3067198", null, "1", "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", "GEO Accession:GSM3067198", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP136369", null, "loader:fastq load.py|options: appendBCtoName platform=Illumina", "DEW160.fastq.gz", "fastq", 958415543.0, 16704238.0, "GSM3067198 r3", "0:57.38", "A:267638469;C:191936701;G:204743895;T:294094008;N:2470", 57, null, null, null, 267638469, 191936701, 204743895, 294094008, 2470, "SRX3841327", "SRS3087487", "SRA672434", "GEO", "Systems Biology, Harvard Medical School", 1, 0.87973, null, 0.12017, null, 0.78922, null, 0.48734, null, 34, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2018-03-23", "Pharyngula", "Embryo", "Embryo Imprecise", "All anatomical structures"], [47795, "SRR6890890", "SRX3841327", "SRS3087487", "SRP136369", "PRJNA445487", "Systematic mapping of cell state trajectories cell lineage and perturbations in the zebrafish embryo using single cell transcriptomics", "GSE112294", "Transcriptome Analysis", "High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease. Here we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach we mapped a cell state landscape that describes axis patterning germ layer formation and organogenesis. We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach \u201cTracerSeq\u201d for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.", null, "pubmed:29700229", null, "TracerSeq Embryo 3", "GSM3067198", null, "tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type", "TracerSeq Embryo 3", "inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al. Nature Protocols 2017 and https://github.com/indrops/. Each sequencing spot is associated with a single biological read and 1 3 technical reads depending on the specific inDrops library preparation chemistry used. FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read. DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read. DEW101 208 used V3 chemistry in which read1 is the biological read read2 carries the first half of the cell barcode read3 carries the library index and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 \"Identify abundant barcodes\" see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1 parameters: n 1 l 15 e 200 m 200 best strata a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads. This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table. UMI counts were adjusted by a total counts normalization. For TracerSeq embryos inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above. These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt. An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv. A table of annotations for each ClusterID. Convert DEW to SRR csv. A table for converting between DEW and NCBI library names. CellTracerCounts csv. A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.", "Zebrafish Embryo Dissociated Cells", null, "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", null, "strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type", "GSM3067198", "GSM3067198: TracerSeq Embryo 3; Danio rerio; RNA Seq", "GSM3067198", null, "1", "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", "GEO Accession:GSM3067198", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP136369", null, "loader:fastq load.py|options: appendBCtoName platform=Illumina", "DEW161.fastq.gz", "fastq", 911486560.0, 15853985.0, "GSM3067198 r4", "0:57.49", "A:254067794;C:182351617;G:196560408;T:278504296;N:2445", 57, null, null, null, 254067794, 182351617, 196560408, 278504296, 2445, "SRX3841327", "SRS3087487", "SRA672434", "GEO", "Systems Biology, Harvard Medical School", 1, 0.88691, null, 0.12363, null, 0.78435, null, 0.50693, null, 60, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2018-03-23", "Pharyngula", "Embryo", "Embryo Imprecise", "All anatomical structures"], [47796, "SRR11699731", "SRX3841327", "SRS3087487", "SRP136369", "PRJNA445487", "Systematic mapping of cell state trajectories cell lineage and perturbations in the zebrafish embryo using single cell transcriptomics", "GSE112294", "Transcriptome Analysis", "High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease. Here we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach we mapped a cell state landscape that describes axis patterning germ layer formation and organogenesis. We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach \u201cTracerSeq\u201d for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.", null, "pubmed:29700229", null, "TracerSeq Embryo 3", "GSM3067198", null, "tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type", "TracerSeq Embryo 3", "inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al. Nature Protocols 2017 and https://github.com/indrops/. Each sequencing spot is associated with a single biological read and 1 3 technical reads depending on the specific inDrops library preparation chemistry used. FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read. DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read. DEW101 208 used V3 chemistry in which read1 is the biological read read2 carries the first half of the cell barcode read3 carries the library index and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 \"Identify abundant barcodes\" see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1 parameters: n 1 l 15 e 200 m 200 best strata a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads. This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table. UMI counts were adjusted by a total counts normalization. For TracerSeq embryos inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above. These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt. An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv. A table of annotations for each ClusterID. Convert DEW to SRR csv. A table for converting between DEW and NCBI library names. CellTracerCounts csv. A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.", "Zebrafish Embryo Dissociated Cells", null, "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", null, "strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type", "GSM3067198", "GSM3067198: TracerSeq Embryo 3; Danio rerio; RNA Seq", "GSM3067198", null, "1", "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", "GEO Accession:GSM3067198", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP136369", null, "loader:fastq load.py|options: appendBCtoName", "DEW158.gfp.fastq.gz", "fastq", 714356770.0, 12690001.0, "GSM3067198 r11", "0:56.29", "A:238801241;C:146515701;G:173416188;T:155618953;N:4687", 56, null, null, null, 238801241, 146515701, 173416188, 155618953, 4687, "SRX3841327", "SRS3087487", "SRA672434", "GEO", "Systems Biology, Harvard Medical School", 1, 0.00355, null, 0.00025, null, 0.99902, null, 0.70542, null, 61, null, "T", null, "under 1.2% mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2018-03-23", "Pharyngula", "Embryo", "Embryo Imprecise", "All anatomical structures"], [47797, "SRR11699732", "SRX3841327", "SRS3087487", "SRP136369", "PRJNA445487", "Systematic mapping of cell state trajectories cell lineage and perturbations in the zebrafish embryo using single cell transcriptomics", "GSE112294", "Transcriptome Analysis", "High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease. Here we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach we mapped a cell state landscape that describes axis patterning germ layer formation and organogenesis. We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach \u201cTracerSeq\u201d for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.", null, "pubmed:29700229", null, "TracerSeq Embryo 3", "GSM3067198", null, "tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type", "TracerSeq Embryo 3", "inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al. Nature Protocols 2017 and https://github.com/indrops/. Each sequencing spot is associated with a single biological read and 1 3 technical reads depending on the specific inDrops library preparation chemistry used. FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read. DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read. DEW101 208 used V3 chemistry in which read1 is the biological read read2 carries the first half of the cell barcode read3 carries the library index and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 \"Identify abundant barcodes\" see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1 parameters: n 1 l 15 e 200 m 200 best strata a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads. This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table. UMI counts were adjusted by a total counts normalization. For TracerSeq embryos inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above. These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt. An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv. A table of annotations for each ClusterID. Convert DEW to SRR csv. A table for converting between DEW and NCBI library names. CellTracerCounts csv. A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.", "Zebrafish Embryo Dissociated Cells", null, "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", null, "strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type", "GSM3067198", "GSM3067198: TracerSeq Embryo 3; Danio rerio; RNA Seq", "GSM3067198", null, "1", "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", "GEO Accession:GSM3067198", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP136369", null, "loader:fastq load.py|options: appendBCtoName", "DEW159.gfp.fastq.gz", "fastq", 692190367.0, 12308538.0, "GSM3067198 r12", "0:56.24", "A:231865817;C:139854334;G:169105319;T:151360408;N:4489", 56, null, null, null, 231865817, 139854334, 169105319, 151360408, 4489, "SRX3841327", "SRS3087487", "SRA672434", "GEO", "Systems Biology, Harvard Medical School", 1, 0.00277, null, 0.00018, null, 0.99931, null, 0.70414, null, 61, null, "T", null, "under 1.2% mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2018-03-23", "Pharyngula", "Embryo", "Embryo Imprecise", "All anatomical structures"], [47798, "SRR11699733", "SRX3841327", "SRS3087487", "SRP136369", "PRJNA445487", "Systematic mapping of cell state trajectories cell lineage and perturbations in the zebrafish embryo using single cell transcriptomics", "GSE112294", "Transcriptome Analysis", "High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease. Here we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach we mapped a cell state landscape that describes axis patterning germ layer formation and organogenesis. We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach \u201cTracerSeq\u201d for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.", null, "pubmed:29700229", null, "TracerSeq Embryo 3", "GSM3067198", null, "tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type", "TracerSeq Embryo 3", "inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al. Nature Protocols 2017 and https://github.com/indrops/. Each sequencing spot is associated with a single biological read and 1 3 technical reads depending on the specific inDrops library preparation chemistry used. FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read. DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read. DEW101 208 used V3 chemistry in which read1 is the biological read read2 carries the first half of the cell barcode read3 carries the library index and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 \"Identify abundant barcodes\" see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1 parameters: n 1 l 15 e 200 m 200 best strata a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads. This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table. UMI counts were adjusted by a total counts normalization. For TracerSeq embryos inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above. These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt. An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv. A table of annotations for each ClusterID. Convert DEW to SRR csv. A table for converting between DEW and NCBI library names. CellTracerCounts csv. A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.", "Zebrafish Embryo Dissociated Cells", null, "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", null, "strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type", "GSM3067198", "GSM3067198: TracerSeq Embryo 3; Danio rerio; RNA Seq", "GSM3067198", null, "1", "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", "GEO Accession:GSM3067198", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP136369", null, "loader:fastq load.py|options: appendBCtoName", "DEW160.gfp.fastq.gz", "fastq", 713049432.0, 12694194.0, "GSM3067198 r13", "0:56.17", "A:239502412;C:145346774;G:172186875;T:156008904;N:4467", 56, null, null, null, 239502412, 145346774, 172186875, 156008904, 4467, "SRX3841327", "SRS3087487", "SRA672434", "GEO", "Systems Biology, Harvard Medical School", 1, 0.00331, null, 0.00023, null, 0.9991, null, 0.70743, null, 61, null, "T", null, "under 1.2% mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2018-03-23", "Pharyngula", "Embryo", "Embryo Imprecise", "All anatomical structures"], [47799, "SRR11699734", "SRX3841327", "SRS3087487", "SRP136369", "PRJNA445487", "Systematic mapping of cell state trajectories cell lineage and perturbations in the zebrafish embryo using single cell transcriptomics", "GSE112294", "Transcriptome Analysis", "High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease. Here we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach we mapped a cell state landscape that describes axis patterning germ layer formation and organogenesis. We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach \u201cTracerSeq\u201d for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.", null, "pubmed:29700229", null, "TracerSeq Embryo 3", "GSM3067198", null, "tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type", "TracerSeq Embryo 3", "inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al. Nature Protocols 2017 and https://github.com/indrops/. Each sequencing spot is associated with a single biological read and 1 3 technical reads depending on the specific inDrops library preparation chemistry used. FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read. DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read. DEW101 208 used V3 chemistry in which read1 is the biological read read2 carries the first half of the cell barcode read3 carries the library index and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 \"Identify abundant barcodes\" see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1 parameters: n 1 l 15 e 200 m 200 best strata a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads. This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table. UMI counts were adjusted by a total counts normalization. For TracerSeq embryos inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above. These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts columns are single cells. Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt. An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv. A table of annotations for each ClusterID. Convert DEW to SRR csv. A table for converting between DEW and NCBI library names. CellTracerCounts csv. A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.", "Zebrafish Embryo Dissociated Cells", null, "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. Nature Protocols 2017", null, "strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type", "GSM3067198", "GSM3067198: TracerSeq Embryo 3; Danio rerio; RNA Seq", "GSM3067198", null, "1", "Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl 2.1 mM KCl 1.2 mM MgSO4 1.8 mM CaNO32 15 mM HEPES pH 7.6]. Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature. Cells were then filtered through a 40\u03bcm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 310g for 5 minutes. Cell pellets were resuspended in 1X DPBS no Ca/Mg Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G and subjected to 2 3 additional rounds of centrifugation and resuspension. post washing cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML. Cell density was quantified manually using INCYTO\u2122 C Chip\u2122 Disposable Hemacytometers Fisher 22 600 100 and adjusted to 100 000 cells per mL. For single embryo dissociations all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature. Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al. Nature Protocols 2017. Following the within droplet reverse transcription step emulsions were split into batches of approximately 1 000 2 000 cells frozen at 80C and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al. 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"sample.accession", "study.accession", "bioproject", "study.title", "study.alias", "study.type", "study.abstract", "study.attributes", "study.PMIDs", "sample.description", "sample.title", "sample.alias", "sample.centername", "sample.attributes", "GEOsample.title", "GEOsample.dataprocessing", "GEOsample.source", "GEOsample.treatmentprotocol", "GEOsample.extractprotocol", "GEOsample.growthprotocol", "GEOsample.characteristics", "GEOsample.accession", "experiment.title", "experiment.alias", "experiment.library_name", "experiment.design_description", "experiment.library_construction_protocol", "experiment.attributes", "experiment.library_strategy", "experiment.library_source", "experiment.library_selection", "experiment.library_layout", "experiment.platform", "experiment.instrument_model", "experiment.spot_descriptor", "experiment.study_ref", "run.title", "run.attributes", "run.filename", "run.semantic_name", "run.total_bases", "run.total_spots", "run.alias", "run.read_lengths", "run.base_counts", "run.r1_length", "run.r2_length", "run.r3_length", "run.r4_length", "run.Acount", "run.Ccount", "run.Gcount", "run.Tcount", "run.Ncount", "run.experiment", "run.pool_member", "submission.accession", "submission.srasource", "submission.bioprojectsource", "seqdetective.n_mates", "seqdetective.mapping_rate.mate1", "seqdetective.mapping_rate.mate2", "seqdetective.nofeature_rate.mate1", "seqdetective.nofeature_rate.mate2", "seqdetective.sparsity.mate1", "seqdetective.sparsity.mate2", "seqdetective.pos_strand_rate.mate1", "seqdetective.pos_strand_rate.mate2", "seqdetective.readlen.mate1", "seqdetective.readlen.mate2", "seqdetective.judgement.mate1", "seqdetective.judgement.mate2", "seqdetective.judgement.reason", "platform_family", "instrument_generation", "read_bias", "selection_class", "prep_kit", "sc_or_bulk", "tech_class", "technology", "tech_variant", "submission.bioprojectsource.country", "earliest_date", "devstage_curation", "devstage_curation_coarse", "tissue_curation", "tissue_curation_coarse"], "primary_keys": [], "units": {}, "query": {"sql": "select rowid, [run.accession], [experiment.accession], [sample.accession], [study.accession], bioproject, [study.title], [study.alias], [study.type], [study.abstract], [study.attributes], [study.PMIDs], [sample.description], [sample.title], [sample.alias], [sample.centername], [sample.attributes], [GEOsample.title], [GEOsample.dataprocessing], [GEOsample.source], [GEOsample.treatmentprotocol], [GEOsample.extractprotocol], [GEOsample.growthprotocol], [GEOsample.characteristics], [GEOsample.accession], [experiment.title], [experiment.alias], [experiment.library_name], [experiment.design_description], [experiment.library_construction_protocol], [experiment.attributes], [experiment.library_strategy], [experiment.library_source], [experiment.library_selection], [experiment.library_layout], [experiment.platform], [experiment.instrument_model], [experiment.spot_descriptor], [experiment.study_ref], [run.title], [run.attributes], [run.filename], [run.semantic_name], [run.total_bases], [run.total_spots], [run.alias], [run.read_lengths], [run.base_counts], [run.r1_length], [run.r2_length], [run.r3_length], [run.r4_length], [run.Acount], [run.Ccount], [run.Gcount], [run.Tcount], [run.Ncount], [run.experiment], [run.pool_member], [submission.accession], [submission.srasource], [submission.bioprojectsource], [seqdetective.n_mates], [seqdetective.mapping_rate.mate1], [seqdetective.mapping_rate.mate2], [seqdetective.nofeature_rate.mate1], [seqdetective.nofeature_rate.mate2], [seqdetective.sparsity.mate1], [seqdetective.sparsity.mate2], [seqdetective.pos_strand_rate.mate1], [seqdetective.pos_strand_rate.mate2], [seqdetective.readlen.mate1], [seqdetective.readlen.mate2], [seqdetective.judgement.mate1], [seqdetective.judgement.mate2], [seqdetective.judgement.reason], platform_family, instrument_generation, read_bias, selection_class, prep_kit, sc_or_bulk, tech_class, technology, tech_variant, [submission.bioprojectsource.country], earliest_date, devstage_curation, devstage_curation_coarse, tissue_curation, tissue_curation_coarse from run_metadata where \"devstage_curation_coarse\" = :p0 and \"technology\" = :p1 order by rowid limit 101", "params": {"p0": "Embryo", "p1": "indrops"}}, "facet_results": {"experiment.library_strategy": {"name": "experiment.library_strategy", "type": "column", "hideable": false, "toggle_url": "/metadata/run_metadata.json?devstage_curation_coarse=Embryo&technology=indrops", "results": [{"value": "RNA-Seq", "label": "RNA-Seq", "count": 94, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?devstage_curation_coarse=Embryo&technology=indrops&experiment.library_strategy=RNA-Seq", "selected": false}, {"value": "OTHER", "label": "OTHER", "count": 58, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?devstage_curation_coarse=Embryo&technology=indrops&experiment.library_strategy=OTHER", "selected": false}], "truncated": false}, "experiment.library_source": {"name": "experiment.library_source", "type": "column", "hideable": false, "toggle_url": "/metadata/run_metadata.json?devstage_curation_coarse=Embryo&technology=indrops", "results": [{"value": "TRANSCRIPTOMIC", "label": "TRANSCRIPTOMIC", "count": 152, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?devstage_curation_coarse=Embryo&technology=indrops&experiment.library_source=TRANSCRIPTOMIC", "selected": false}], "truncated": false}, "experiment.library_selection": {"name": "experiment.library_selection", "type": "column", "hideable": false, "toggle_url": "/metadata/run_metadata.json?devstage_curation_coarse=Embryo&technology=indrops", "results": [{"value": "cDNA", "label": "cDNA", "count": 94, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?devstage_curation_coarse=Embryo&technology=indrops&experiment.library_selection=cDNA", "selected": false}, {"value": "other", "label": "other", "count": 58, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?devstage_curation_coarse=Embryo&technology=indrops&experiment.library_selection=other", "selected": false}], "truncated": false}, "experiment.library_layout": {"name": "experiment.library_layout", "type": "column", "hideable": false, "toggle_url": "/metadata/run_metadata.json?devstage_curation_coarse=Embryo&technology=indrops", "results": [{"value": "SINGLE", "label": "SINGLE", "count": 94, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?devstage_curation_coarse=Embryo&technology=indrops&experiment.library_layout=SINGLE", "selected": false}, {"value": "PAIRED", "label": "PAIRED", "count": 58, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?devstage_curation_coarse=Embryo&technology=indrops&experiment.library_layout=PAIRED", "selected": false}], "truncated": false}, "experiment.platform": {"name": "experiment.platform", "type": "column", "hideable": false, "toggle_url": "/metadata/run_metadata.json?devstage_curation_coarse=Embryo&technology=indrops", "results": [{"value": "ILLUMINA", "label": "ILLUMINA", "count": 152, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?devstage_curation_coarse=Embryo&technology=indrops&experiment.platform=ILLUMINA", "selected": false}], "truncated": false}, "devstage_curation_coarse": {"name": "devstage_curation_coarse", "type": "column", "hideable": false, "toggle_url": "/metadata/run_metadata.json?devstage_curation_coarse=Embryo&technology=indrops", "results": [{"value": "Embryo", "label": "Embryo", "count": 152, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?technology=indrops", "selected": true}], "truncated": false}, "devstage_curation": {"name": "devstage_curation", "type": "column", "hideable": false, "toggle_url": "/metadata/run_metadata.json?devstage_curation_coarse=Embryo&technology=indrops", "results": [{"value": "Multi-stage", "label": "Multi-stage", "count": 58, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?devstage_curation_coarse=Embryo&technology=indrops&devstage_curation=Multi-stage", "selected": false}, {"value": "Pharyngula", "label": "Pharyngula", "count": 51, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?devstage_curation_coarse=Embryo&technology=indrops&devstage_curation=Pharyngula", "selected": false}, {"value": "Segmentation", "label": "Segmentation", "count": 29, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?devstage_curation_coarse=Embryo&technology=indrops&devstage_curation=Segmentation", "selected": false}, {"value": "Gastrula", "label": "Gastrula", "count": 12, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?devstage_curation_coarse=Embryo&technology=indrops&devstage_curation=Gastrula", "selected": false}, {"value": "Blastula", "label": "Blastula", "count": 2, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?devstage_curation_coarse=Embryo&technology=indrops&devstage_curation=Blastula", "selected": false}], "truncated": false}, "tissue_curation_coarse": {"name": "tissue_curation_coarse", "type": "column", "hideable": false, "toggle_url": "/metadata/run_metadata.json?devstage_curation_coarse=Embryo&technology=indrops", "results": [{"value": "All anatomical structures", "label": "All anatomical structures", "count": 150, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?devstage_curation_coarse=Embryo&technology=indrops&tissue_curation_coarse=All+anatomical+structures", "selected": false}, {"value": "Multi-system", "label": "Multi-system", "count": 2, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?devstage_curation_coarse=Embryo&technology=indrops&tissue_curation_coarse=Multi-system", "selected": false}], "truncated": false}, "tissue_curation": {"name": "tissue_curation", "type": "column", "hideable": false, "toggle_url": "/metadata/run_metadata.json?devstage_curation_coarse=Embryo&technology=indrops", "results": [{"value": "Embryo Imprecise", "label": "Embryo Imprecise", "count": 94, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?devstage_curation_coarse=Embryo&technology=indrops&tissue_curation=Embryo+Imprecise", "selected": false}, {"value": "Whole Organism", "label": "Whole Organism", "count": 56, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?devstage_curation_coarse=Embryo&technology=indrops&tissue_curation=Whole+Organism", "selected": false}, {"value": "Tail", "label": "Tail", "count": 2, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?devstage_curation_coarse=Embryo&technology=indrops&tissue_curation=Tail", "selected": false}], "truncated": false}, "technology": {"name": "technology", "type": "column", "hideable": false, "toggle_url": "/metadata/run_metadata.json?devstage_curation_coarse=Embryo&technology=indrops", "results": [{"value": "indrops", "label": "indrops", "count": 152, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?devstage_curation_coarse=Embryo", "selected": true}], "truncated": false}}, "suggested_facets": [], "next": "47799", "next_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?devstage_curation_coarse=Embryo&technology=indrops&_next=47799", "private": false, "allow_execute_sql": true, "query_ms": 103.79968599590939}