{"database": "metadata", "table": "run_metadata", "is_view": false, "human_description_en": "where experiment.library_source = \"TRANSCRIPTOMIC\" 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"], [42599, "SRR5810682", "SRX2989237", "SRS2341160", "SRP111339", "PRJNA393429", "Dissecting hematopoietic and renal cell heterogeneity in adult zebrafish at single cell resolution using RNA sequencing [inDrops]", "GSE100910", "Transcriptome Analysis", "Recent advances in single cell transcriptomic profiling have provided unprecedented access to investigate cell heterogeneity during tissue and organ development. Here we utilized massively parallel single cell RNA sequencing to define cell heterogeneity within the zebrafish kidney marrow constructing a comprehensive molecular atlas of definitive hematopoiesis and functionally distinct renal cells found in adult zebrafish. Because our method analyzed blood and kidney cells in an unbiased manner our approach was useful in characterizing immune cell deficiencies within prkdcD3612fs il2rgaY91fs and double homozygous mutant fish identifying blood cell losses in T B and natural killer cells within specific genetic mutants. Our analysis also uncovered novel cell types including two classes of natural killer immune cells classically defined and erythroid primed hematopoietic stem and progenitor cells mucin secreting kidney cells and kidney stem/progenitor cells. In total our work provides the first comprehensive single cell transcriptomic analysis of kidney and marrow cells in the adult zebrafish. Overall design: The goal of our study is to establish the transcriptional profiles of hematopoietic and kidney cell lineages residing in the zebrafish whole kidney marrow. Firstly we performed single cell RNA sequencing by a modified Smart seq2 protocol on sorted single cells from fluorescent transgenic zebrafish lines which label distinct blood cell types n = 246 cells total. Secondly we utilized droplet based single cell RNA sequencing inDrop to investigate unmarked comprehensive hematopoietic lineage structure within wild type casper strain zebrafish N=3 animals n=3 782 cells total. From this we identified ten distinct hematopoietic groups of blood and immune identities. Thirdly we confirmed blood lineage interpretations by comparing hematopoietic lineages within wild type fish with mutant zebrafish with known immunodeficiencies including prkdcD3612fs N=3 animals n=3 201 cells il2rgaY91fs N=2 animals n=2 068 cells and prkdcD3612fs il2rgaY91fs double compound mutant fish N=2 animals n=2 276 cells. Lastly we identified seven structural and functional cell lineages of kidney identities in the whole kidney marrow n=1 699 kidney cells.", "parent bioproject:PRJNA393415", "pubmed:28878000", null, "inDrop sequencing of multiple samples 2", "GSM2696104", null, "source name:Whole kidney marrow|indrop version:inDrop V3 protocol|tissue:kidney marrow|genotype:multiple genotypes", "inDrop sequencing of multiple samples 2", "Reads were processed using the indrops pipelinehttps://github.com/indrops/indrops. Briefly reads were filtered according to structure and quality criteria. Filtered reads were then they were sorted by barcode. Demultiplexed reads were aligned to the GRCz10 transcriptome using Bowtie. Please see file \"inDrop GEO supplementary.xlsx \" available on the series record for information on de multiplexing and barcodes for individual samples and cells. Genome build: GRCz10 Supplementary files format and content: text file with read counts.", "Whole kidney marrow", null, "RNA from individual cells were reverse transcribed barcoded and unique transcripts are imprinted with UMI all within the droplet. RNA from 1 500 cells of each animal were pooled and subsequently processed together. cDNA products were pre amplified to produce a cDNA library compatible with the Illumina Nextseq platform. Three sequencing runs were completed using the Nextseq 500 High Output V2 kit 75 cycles Illumina on a NextSeq 500 platform Illumina. Zebrafish cells are isolated from the kidney marrow into single cell suspensions and treated with 15% Optiprep Sigma. Single cells are microfluidically sorted into droplets containing reverse transcription and barcoding reagents. This method is outlined in Klein et al. PMID 26000487 and Zilionis et al. PMID 27929523.", null, "indrop version:inDrop V3 protocol|tissue:kidney marrow|genotype:multiple genotypes", "GSM2696104", "GSM2696104: inDrop sequencing of multiple samples 2; Danio rerio; RNA Seq", "GSM2696104", null, "1", "RNA from individual cells were reverse transcribed barcoded and unique transcripts are imprinted with UMI all within the droplet. RNA from 1 500 cells of each animal were pooled and subsequently processed together. cDNA products were pre amplified to produce a cDNA library compatible with the Illumina Nextseq platform. Three sequencing runs were completed using the Nextseq 500 High Output V2 kit 75 cycles Illumina on a NextSeq 500 platform Illumina. Zebrafish cells are isolated from the kidney marrow into single cell suspensions and treated with 15% Optiprep Sigma. Single cells are microfluidically sorted into droplets containing reverse transcription and barcoding reagents. This method is outlined in Klein et al. PMID 26000487 and Zilionis et al. PMID 27929523.", "GEO Accession:GSM2696104", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP111339", null, null, "inDrop_v3_run2_R1.fastq.gz", "fastq", 23315853588.0, 382227108.0, "GSM2696104 r1", "0:61 1:0", "A:6573828142;C:5137891481;G:5021324431;T:6582457296;N:352238", 61, 0, null, null, 6573828142, 5137891481, 5021324431, 6582457296, 352238, "SRX2989237", "SRS2341160", "SRA584587", "GEO", "Pathology, Massachusetts General Hospital", 1, 0.83828, null, 0.08537, null, 0.83552, null, 0.56658, null, 61, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "random_priming", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-07-07", "Undetermined", "Adult", "Kidney", "Renal System"], [42600, "SRR5810683", "SRX2989237", "SRS2341160", "SRP111339", "PRJNA393429", "Dissecting hematopoietic and renal cell heterogeneity in adult zebrafish at single cell resolution using RNA sequencing [inDrops]", "GSE100910", "Transcriptome Analysis", "Recent advances in single cell transcriptomic profiling have provided unprecedented access to investigate cell heterogeneity during tissue and organ development. Here we utilized massively parallel single cell RNA sequencing to define cell heterogeneity within the zebrafish kidney marrow constructing a comprehensive molecular atlas of definitive hematopoiesis and functionally distinct renal cells found in adult zebrafish. Because our method analyzed blood and kidney cells in an unbiased manner our approach was useful in characterizing immune cell deficiencies within prkdcD3612fs il2rgaY91fs and double homozygous mutant fish identifying blood cell losses in T B and natural killer cells within specific genetic mutants. Our analysis also uncovered novel cell types including two classes of natural killer immune cells classically defined and erythroid primed hematopoietic stem and progenitor cells mucin secreting kidney cells and kidney stem/progenitor cells. In total our work provides the first comprehensive single cell transcriptomic analysis of kidney and marrow cells in the adult zebrafish. Overall design: The goal of our study is to establish the transcriptional profiles of hematopoietic and kidney cell lineages residing in the zebrafish whole kidney marrow. Firstly we performed single cell RNA sequencing by a modified Smart seq2 protocol on sorted single cells from fluorescent transgenic zebrafish lines which label distinct blood cell types n = 246 cells total. Secondly we utilized droplet based single cell RNA sequencing inDrop to investigate unmarked comprehensive hematopoietic lineage structure within wild type casper strain zebrafish N=3 animals n=3 782 cells total. From this we identified ten distinct hematopoietic groups of blood and immune identities. Thirdly we confirmed blood lineage interpretations by comparing hematopoietic lineages within wild type fish with mutant zebrafish with known immunodeficiencies including prkdcD3612fs N=3 animals n=3 201 cells il2rgaY91fs N=2 animals n=2 068 cells and prkdcD3612fs il2rgaY91fs double compound mutant fish N=2 animals n=2 276 cells. Lastly we identified seven structural and functional cell lineages of kidney identities in the whole kidney marrow n=1 699 kidney cells.", "parent bioproject:PRJNA393415", "pubmed:28878000", null, "inDrop sequencing of multiple samples 2", "GSM2696104", null, "source name:Whole kidney marrow|indrop version:inDrop V3 protocol|tissue:kidney marrow|genotype:multiple genotypes", "inDrop sequencing of multiple samples 2", "Reads were processed using the indrops pipelinehttps://github.com/indrops/indrops. Briefly reads were filtered according to structure and quality criteria. Filtered reads were then they were sorted by barcode. Demultiplexed reads were aligned to the GRCz10 transcriptome using Bowtie. Please see file \"inDrop GEO supplementary.xlsx \" available on the series record for information on de multiplexing and barcodes for individual samples and cells. Genome build: GRCz10 Supplementary files format and content: text file with read counts.", "Whole kidney marrow", null, "RNA from individual cells were reverse transcribed barcoded and unique transcripts are imprinted with UMI all within the droplet. RNA from 1 500 cells of each animal were pooled and subsequently processed together. cDNA products were pre amplified to produce a cDNA library compatible with the Illumina Nextseq platform. Three sequencing runs were completed using the Nextseq 500 High Output V2 kit 75 cycles Illumina on a NextSeq 500 platform Illumina. Zebrafish cells are isolated from the kidney marrow into single cell suspensions and treated with 15% Optiprep Sigma. Single cells are microfluidically sorted into droplets containing reverse transcription and barcoding reagents. This method is outlined in Klein et al. PMID 26000487 and Zilionis et al. PMID 27929523.", null, "indrop version:inDrop V3 protocol|tissue:kidney marrow|genotype:multiple genotypes", "GSM2696104", "GSM2696104: inDrop sequencing of multiple samples 2; Danio rerio; RNA Seq", "GSM2696104", null, "1", "RNA from individual cells were reverse transcribed barcoded and unique transcripts are imprinted with UMI all within the droplet. RNA from 1 500 cells of each animal were pooled and subsequently processed together. cDNA products were pre amplified to produce a cDNA library compatible with the Illumina Nextseq platform. Three sequencing runs were completed using the Nextseq 500 High Output V2 kit 75 cycles Illumina on a NextSeq 500 platform Illumina. Zebrafish cells are isolated from the kidney marrow into single cell suspensions and treated with 15% Optiprep Sigma. Single cells are microfluidically sorted into droplets containing reverse transcription and barcoding reagents. This method is outlined in Klein et al. PMID 26000487 and Zilionis et al. PMID 27929523.", "GEO Accession:GSM2696104", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP111339", null, null, "inDrop_v3_run2_R2.fastq.gz", "fastq", 3057816864.0, 382227108.0, "GSM2696104 r2", "0:0 1:8", "A:743619897;C:720488231;G:1031094190;T:562297818;N:316728", 0, 8, null, null, 743619897, 720488231, 1031094190, 562297818, 316728, "SRX2989237", "SRS2341160", "SRA584587", "GEO", "Pathology, Massachusetts General Hospital", 1, 0.0, null, 0.0, null, 1.0, null, null, null, 8, null, "T", null, "under 1.2% mapping rate", "illumina", "nextseq", "unknown", "random_priming", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-07-07", "Undetermined", "Adult", "Kidney", "Renal System"], [42601, "SRR5810684", "SRX2989237", "SRS2341160", "SRP111339", "PRJNA393429", "Dissecting hematopoietic and renal cell heterogeneity in adult zebrafish at single cell resolution using RNA sequencing [inDrops]", "GSE100910", "Transcriptome Analysis", "Recent advances in single cell transcriptomic profiling have provided unprecedented access to investigate cell heterogeneity during tissue and organ development. Here we utilized massively parallel single cell RNA sequencing to define cell heterogeneity within the zebrafish kidney marrow constructing a comprehensive molecular atlas of definitive hematopoiesis and functionally distinct renal cells found in adult zebrafish. Because our method analyzed blood and kidney cells in an unbiased manner our approach was useful in characterizing immune cell deficiencies within prkdcD3612fs il2rgaY91fs and double homozygous mutant fish identifying blood cell losses in T B and natural killer cells within specific genetic mutants. Our analysis also uncovered novel cell types including two classes of natural killer immune cells classically defined and erythroid primed hematopoietic stem and progenitor cells mucin secreting kidney cells and kidney stem/progenitor cells. In total our work provides the first comprehensive single cell transcriptomic analysis of kidney and marrow cells in the adult zebrafish. Overall design: The goal of our study is to establish the transcriptional profiles of hematopoietic and kidney cell lineages residing in the zebrafish whole kidney marrow. Firstly we performed single cell RNA sequencing by a modified Smart seq2 protocol on sorted single cells from fluorescent transgenic zebrafish lines which label distinct blood cell types n = 246 cells total. Secondly we utilized droplet based single cell RNA sequencing inDrop to investigate unmarked comprehensive hematopoietic lineage structure within wild type casper strain zebrafish N=3 animals n=3 782 cells total. From this we identified ten distinct hematopoietic groups of blood and immune identities. Thirdly we confirmed blood lineage interpretations by comparing hematopoietic lineages within wild type fish with mutant zebrafish with known immunodeficiencies including prkdcD3612fs N=3 animals n=3 201 cells il2rgaY91fs N=2 animals n=2 068 cells and prkdcD3612fs il2rgaY91fs double compound mutant fish N=2 animals n=2 276 cells. Lastly we identified seven structural and functional cell lineages of kidney identities in the whole kidney marrow n=1 699 kidney cells.", "parent bioproject:PRJNA393415", "pubmed:28878000", null, "inDrop sequencing of multiple samples 2", "GSM2696104", null, "source name:Whole kidney marrow|indrop version:inDrop V3 protocol|tissue:kidney marrow|genotype:multiple genotypes", "inDrop sequencing of multiple samples 2", "Reads were processed using the indrops pipelinehttps://github.com/indrops/indrops. Briefly reads were filtered according to structure and quality criteria. Filtered reads were then they were sorted by barcode. Demultiplexed reads were aligned to the GRCz10 transcriptome using Bowtie. Please see file \"inDrop GEO supplementary.xlsx \" available on the series record for information on de multiplexing and barcodes for individual samples and cells. Genome build: GRCz10 Supplementary files format and content: text file with read counts.", "Whole kidney marrow", null, "RNA from individual cells were reverse transcribed barcoded and unique transcripts are imprinted with UMI all within the droplet. RNA from 1 500 cells of each animal were pooled and subsequently processed together. cDNA products were pre amplified to produce a cDNA library compatible with the Illumina Nextseq platform. Three sequencing runs were completed using the Nextseq 500 High Output V2 kit 75 cycles Illumina on a NextSeq 500 platform Illumina. Zebrafish cells are isolated from the kidney marrow into single cell suspensions and treated with 15% Optiprep Sigma. Single cells are microfluidically sorted into droplets containing reverse transcription and barcoding reagents. This method is outlined in Klein et al. PMID 26000487 and Zilionis et al. PMID 27929523.", null, "indrop version:inDrop V3 protocol|tissue:kidney marrow|genotype:multiple genotypes", "GSM2696104", "GSM2696104: inDrop sequencing of multiple samples 2; Danio rerio; RNA Seq", "GSM2696104", null, "1", "RNA from individual cells were reverse transcribed barcoded and unique transcripts are imprinted with UMI all within the droplet. RNA from 1 500 cells of each animal were pooled and subsequently processed together. cDNA products were pre amplified to produce a cDNA library compatible with the Illumina Nextseq platform. Three sequencing runs were completed using the Nextseq 500 High Output V2 kit 75 cycles Illumina on a NextSeq 500 platform Illumina. Zebrafish cells are isolated from the kidney marrow into single cell suspensions and treated with 15% Optiprep Sigma. Single cells are microfluidically sorted into droplets containing reverse transcription and barcoding reagents. This method is outlined in Klein et al. PMID 26000487 and Zilionis et al. PMID 27929523.", "GEO Accession:GSM2696104", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP111339", null, null, "inDrop_v3_run2_R3.fastq.gz", "fastq", 3057816864.0, 382227108.0, "GSM2696104 r3", "0:8", "A:913664473;C:577895371;G:646194239;T:918893384;N:1169397", 8, null, null, null, 913664473, 577895371, 646194239, 918893384, 1169397, "SRX2989237", "SRS2341160", "SRA584587", "GEO", "Pathology, Massachusetts General Hospital", 1, 0.0, null, 0.0, null, 1.0, null, null, null, 8, null, "T", null, "under 1.2% mapping rate", "illumina", "nextseq", "unknown", "random_priming", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-07-07", "Undetermined", "Adult", "Kidney", "Renal System"], [42602, "SRR5810685", "SRX2989237", "SRS2341160", "SRP111339", "PRJNA393429", "Dissecting hematopoietic and renal cell heterogeneity in adult zebrafish at single cell resolution using RNA sequencing [inDrops]", "GSE100910", "Transcriptome Analysis", "Recent advances in single cell transcriptomic profiling have provided unprecedented access to investigate cell heterogeneity during tissue and organ development. Here we utilized massively parallel single cell RNA sequencing to define cell heterogeneity within the zebrafish kidney marrow constructing a comprehensive molecular atlas of definitive hematopoiesis and functionally distinct renal cells found in adult zebrafish. Because our method analyzed blood and kidney cells in an unbiased manner our approach was useful in characterizing immune cell deficiencies within prkdcD3612fs il2rgaY91fs and double homozygous mutant fish identifying blood cell losses in T B and natural killer cells within specific genetic mutants. Our analysis also uncovered novel cell types including two classes of natural killer immune cells classically defined and erythroid primed hematopoietic stem and progenitor cells mucin secreting kidney cells and kidney stem/progenitor cells. In total our work provides the first comprehensive single cell transcriptomic analysis of kidney and marrow cells in the adult zebrafish. Overall design: The goal of our study is to establish the transcriptional profiles of hematopoietic and kidney cell lineages residing in the zebrafish whole kidney marrow. Firstly we performed single cell RNA sequencing by a modified Smart seq2 protocol on sorted single cells from fluorescent transgenic zebrafish lines which label distinct blood cell types n = 246 cells total. Secondly we utilized droplet based single cell RNA sequencing inDrop to investigate unmarked comprehensive hematopoietic lineage structure within wild type casper strain zebrafish N=3 animals n=3 782 cells total. From this we identified ten distinct hematopoietic groups of blood and immune identities. Thirdly we confirmed blood lineage interpretations by comparing hematopoietic lineages within wild type fish with mutant zebrafish with known immunodeficiencies including prkdcD3612fs N=3 animals n=3 201 cells il2rgaY91fs N=2 animals n=2 068 cells and prkdcD3612fs il2rgaY91fs double compound mutant fish N=2 animals n=2 276 cells. Lastly we identified seven structural and functional cell lineages of kidney identities in the whole kidney marrow n=1 699 kidney cells.", "parent bioproject:PRJNA393415", "pubmed:28878000", null, "inDrop sequencing of multiple samples 2", "GSM2696104", null, "source name:Whole kidney marrow|indrop version:inDrop V3 protocol|tissue:kidney marrow|genotype:multiple genotypes", "inDrop sequencing of multiple samples 2", "Reads were processed using the indrops pipelinehttps://github.com/indrops/indrops. Briefly reads were filtered according to structure and quality criteria. Filtered reads were then they were sorted by barcode. Demultiplexed reads were aligned to the GRCz10 transcriptome using Bowtie. Please see file \"inDrop GEO supplementary.xlsx \" available on the series record for information on de multiplexing and barcodes for individual samples and cells. Genome build: GRCz10 Supplementary files format and content: text file with read counts.", "Whole kidney marrow", null, "RNA from individual cells were reverse transcribed barcoded and unique transcripts are imprinted with UMI all within the droplet. RNA from 1 500 cells of each animal were pooled and subsequently processed together. cDNA products were pre amplified to produce a cDNA library compatible with the Illumina Nextseq platform. Three sequencing runs were completed using the Nextseq 500 High Output V2 kit 75 cycles Illumina on a NextSeq 500 platform Illumina. Zebrafish cells are isolated from the kidney marrow into single cell suspensions and treated with 15% Optiprep Sigma. Single cells are microfluidically sorted into droplets containing reverse transcription and barcoding reagents. This method is outlined in Klein et al. PMID 26000487 and Zilionis et al. PMID 27929523.", null, "indrop version:inDrop V3 protocol|tissue:kidney marrow|genotype:multiple genotypes", "GSM2696104", "GSM2696104: inDrop sequencing of multiple samples 2; Danio rerio; RNA Seq", "GSM2696104", null, "1", "RNA from individual cells were reverse transcribed barcoded and unique transcripts are imprinted with UMI all within the droplet. RNA from 1 500 cells of each animal were pooled and subsequently processed together. cDNA products were pre amplified to produce a cDNA library compatible with the Illumina Nextseq platform. Three sequencing runs were completed using the Nextseq 500 High Output V2 kit 75 cycles Illumina on a NextSeq 500 platform Illumina. Zebrafish cells are isolated from the kidney marrow into single cell suspensions and treated with 15% Optiprep Sigma. Single cells are microfluidically sorted into droplets containing reverse transcription and barcoding reagents. This method is outlined in Klein et al. PMID 26000487 and Zilionis et al. PMID 27929523.", "GEO Accession:GSM2696104", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP111339", null, null, "inDrop_v3_run2_R4.fastq.gz", "fastq", 5351179512.0, 382227108.0, "GSM2696104 r4", "0:14", "A:1351470715;C:1294400719;G:1379073889;T:1322397488;N:3836701", 14, null, null, null, 1351470715, 1294400719, 1379073889, 1322397488, 3836701, "SRX2989237", "SRS2341160", "SRA584587", "GEO", "Pathology, Massachusetts General Hospital", 1, 0.0, null, 0.0, null, 1.0, null, null, null, 14, null, "T", null, "under 1.2% mapping rate", "illumina", "nextseq", "unknown", "random_priming", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-07-07", "Undetermined", "Adult", "Kidney", "Renal System"], [42603, "SRR5810678", "SRX2989236", "SRS2341159", "SRP111339", "PRJNA393429", "Dissecting hematopoietic and renal cell heterogeneity in adult zebrafish at single cell resolution using RNA sequencing [inDrops]", "GSE100910", "Transcriptome Analysis", "Recent advances in single cell transcriptomic profiling have provided unprecedented access to investigate cell heterogeneity during tissue and organ development. Here we utilized massively parallel single cell RNA sequencing to define cell heterogeneity within the zebrafish kidney marrow constructing a comprehensive molecular atlas of definitive hematopoiesis and functionally distinct renal cells found in adult zebrafish. Because our method analyzed blood and kidney cells in an unbiased manner our approach was useful in characterizing immune cell deficiencies within prkdcD3612fs il2rgaY91fs and double homozygous mutant fish identifying blood cell losses in T B and natural killer cells within specific genetic mutants. Our analysis also uncovered novel cell types including two classes of natural killer immune cells classically defined and erythroid primed hematopoietic stem and progenitor cells mucin secreting kidney cells and kidney stem/progenitor cells. In total our work provides the first comprehensive single cell transcriptomic analysis of kidney and marrow cells in the adult zebrafish. Overall design: The goal of our study is to establish the transcriptional profiles of hematopoietic and kidney cell lineages residing in the zebrafish whole kidney marrow. Firstly we performed single cell RNA sequencing by a modified Smart seq2 protocol on sorted single cells from fluorescent transgenic zebrafish lines which label distinct blood cell types n = 246 cells total. Secondly we utilized droplet based single cell RNA sequencing inDrop to investigate unmarked comprehensive hematopoietic lineage structure within wild type casper strain zebrafish N=3 animals n=3 782 cells total. From this we identified ten distinct hematopoietic groups of blood and immune identities. Thirdly we confirmed blood lineage interpretations by comparing hematopoietic lineages within wild type fish with mutant zebrafish with known immunodeficiencies including prkdcD3612fs N=3 animals n=3 201 cells il2rgaY91fs N=2 animals n=2 068 cells and prkdcD3612fs il2rgaY91fs double compound mutant fish N=2 animals n=2 276 cells. Lastly we identified seven structural and functional cell lineages of kidney identities in the whole kidney marrow n=1 699 kidney cells.", "parent bioproject:PRJNA393415", "pubmed:28878000", null, "inDrop sequencing of multiple samples 1", "GSM2696103", null, "source name:Whole kidney marrow|indrop version:inDrop V3 protocol|tissue:kidney marrow|genotype:multiple genotypes", "inDrop sequencing of multiple samples 1", "Reads were processed using the indrops pipelinehttps://github.com/indrops/indrops. Briefly reads were filtered according to structure and quality criteria. Filtered reads were then they were sorted by barcode. Demultiplexed reads were aligned to the GRCz10 transcriptome using Bowtie. Please see file \"inDrop GEO supplementary.xlsx \" available on the series record for information on de multiplexing and barcodes for individual samples and cells. Genome build: GRCz10 Supplementary files format and content: text file with read counts.", "Whole kidney marrow", null, "RNA from individual cells were reverse transcribed barcoded and unique transcripts are imprinted with UMI all within the droplet. RNA from 1 500 cells of each animal were pooled and subsequently processed together. cDNA products were pre amplified to produce a cDNA library compatible with the Illumina Nextseq platform. Three sequencing runs were completed using the Nextseq 500 High Output V2 kit 75 cycles Illumina on a NextSeq 500 platform Illumina. Zebrafish cells are isolated from the kidney marrow into single cell suspensions and treated with 15% Optiprep Sigma. Single cells are microfluidically sorted into droplets containing reverse transcription and barcoding reagents. This method is outlined in Klein et al. PMID 26000487 and Zilionis et al. PMID 27929523.", null, "indrop version:inDrop V3 protocol|tissue:kidney marrow|genotype:multiple genotypes", "GSM2696103", "GSM2696103: inDrop sequencing of multiple samples 1; Danio rerio; RNA Seq", "GSM2696103", null, "1", "RNA from individual cells were reverse transcribed barcoded and unique transcripts are imprinted with UMI all within the droplet. RNA from 1 500 cells of each animal were pooled and subsequently processed together. cDNA products were pre amplified to produce a cDNA library compatible with the Illumina Nextseq platform. Three sequencing runs were completed using the Nextseq 500 High Output V2 kit 75 cycles Illumina on a NextSeq 500 platform Illumina. Zebrafish cells are isolated from the kidney marrow into single cell suspensions and treated with 15% Optiprep Sigma. Single cells are microfluidically sorted into droplets containing reverse transcription and barcoding reagents. This method is outlined in Klein et al. PMID 26000487 and Zilionis et al. PMID 27929523.", "GEO Accession:GSM2696103", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP111339", null, null, "inDrop_v3_run1_R1.fastq.gz", "fastq", 26868371039.0, 440465099.0, "GSM2696103 r1", "0:61 1:0", "A:7598044404;C:5948542485;G:5746339343;T:7575168754;N:276053", 61, 0, null, null, 7598044404, 5948542485, 5746339343, 7575168754, 276053, "SRX2989236", "SRS2341159", "SRA584587", "GEO", "Pathology, Massachusetts General Hospital", 1, 0.83437, null, 0.08222, null, 0.82658, null, 0.54569, null, 61, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "random_priming", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-07-07", "Undetermined", "Adult", "Kidney", "Renal System"], [42604, "SRR5810679", "SRX2989236", "SRS2341159", "SRP111339", "PRJNA393429", "Dissecting hematopoietic and renal cell heterogeneity in adult zebrafish at single cell resolution using RNA sequencing [inDrops]", "GSE100910", "Transcriptome Analysis", "Recent advances in single cell transcriptomic profiling have provided unprecedented access to investigate cell heterogeneity during tissue and organ development. Here we utilized massively parallel single cell RNA sequencing to define cell heterogeneity within the zebrafish kidney marrow constructing a comprehensive molecular atlas of definitive hematopoiesis and functionally distinct renal cells found in adult zebrafish. Because our method analyzed blood and kidney cells in an unbiased manner our approach was useful in characterizing immune cell deficiencies within prkdcD3612fs il2rgaY91fs and double homozygous mutant fish identifying blood cell losses in T B and natural killer cells within specific genetic mutants. Our analysis also uncovered novel cell types including two classes of natural killer immune cells classically defined and erythroid primed hematopoietic stem and progenitor cells mucin secreting kidney cells and kidney stem/progenitor cells. In total our work provides the first comprehensive single cell transcriptomic analysis of kidney and marrow cells in the adult zebrafish. Overall design: The goal of our study is to establish the transcriptional profiles of hematopoietic and kidney cell lineages residing in the zebrafish whole kidney marrow. Firstly we performed single cell RNA sequencing by a modified Smart seq2 protocol on sorted single cells from fluorescent transgenic zebrafish lines which label distinct blood cell types n = 246 cells total. Secondly we utilized droplet based single cell RNA sequencing inDrop to investigate unmarked comprehensive hematopoietic lineage structure within wild type casper strain zebrafish N=3 animals n=3 782 cells total. From this we identified ten distinct hematopoietic groups of blood and immune identities. Thirdly we confirmed blood lineage interpretations by comparing hematopoietic lineages within wild type fish with mutant zebrafish with known immunodeficiencies including prkdcD3612fs N=3 animals n=3 201 cells il2rgaY91fs N=2 animals n=2 068 cells and prkdcD3612fs il2rgaY91fs double compound mutant fish N=2 animals n=2 276 cells. Lastly we identified seven structural and functional cell lineages of kidney identities in the whole kidney marrow n=1 699 kidney cells.", "parent bioproject:PRJNA393415", "pubmed:28878000", null, "inDrop sequencing of multiple samples 1", "GSM2696103", null, "source name:Whole kidney marrow|indrop version:inDrop V3 protocol|tissue:kidney marrow|genotype:multiple genotypes", "inDrop sequencing of multiple samples 1", "Reads were processed using the indrops pipelinehttps://github.com/indrops/indrops. Briefly reads were filtered according to structure and quality criteria. Filtered reads were then they were sorted by barcode. Demultiplexed reads were aligned to the GRCz10 transcriptome using Bowtie. Please see file \"inDrop GEO supplementary.xlsx \" available on the series record for information on de multiplexing and barcodes for individual samples and cells. Genome build: GRCz10 Supplementary files format and content: text file with read counts.", "Whole kidney marrow", null, "RNA from individual cells were reverse transcribed barcoded and unique transcripts are imprinted with UMI all within the droplet. RNA from 1 500 cells of each animal were pooled and subsequently processed together. cDNA products were pre amplified to produce a cDNA library compatible with the Illumina Nextseq platform. Three sequencing runs were completed using the Nextseq 500 High Output V2 kit 75 cycles Illumina on a NextSeq 500 platform Illumina. Zebrafish cells are isolated from the kidney marrow into single cell suspensions and treated with 15% Optiprep Sigma. Single cells are microfluidically sorted into droplets containing reverse transcription and barcoding reagents. This method is outlined in Klein et al. PMID 26000487 and Zilionis et al. PMID 27929523.", null, "indrop version:inDrop V3 protocol|tissue:kidney marrow|genotype:multiple genotypes", "GSM2696103", "GSM2696103: inDrop sequencing of multiple samples 1; Danio rerio; RNA Seq", "GSM2696103", null, "1", "RNA from individual cells were reverse transcribed barcoded and unique transcripts are imprinted with UMI all within the droplet. RNA from 1 500 cells of each animal were pooled and subsequently processed together. cDNA products were pre amplified to produce a cDNA library compatible with the Illumina Nextseq platform. Three sequencing runs were completed using the Nextseq 500 High Output V2 kit 75 cycles Illumina on a NextSeq 500 platform Illumina. Zebrafish cells are isolated from the kidney marrow into single cell suspensions and treated with 15% Optiprep Sigma. Single cells are microfluidically sorted into droplets containing reverse transcription and barcoding reagents. This method is outlined in Klein et al. PMID 26000487 and Zilionis et al. PMID 27929523.", "GEO Accession:GSM2696103", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP111339", null, null, "inDrop_v3_run1_R2.fastq.gz", "fastq", 3523720792.0, 440465099.0, "GSM2696103 r2", "0:0 1:8", "A:892841722;C:781074758;G:1176856476;T:672376743;N:571093", 0, 8, null, null, 892841722, 781074758, 1176856476, 672376743, 571093, "SRX2989236", "SRS2341159", "SRA584587", "GEO", "Pathology, Massachusetts General Hospital", 1, 0.0, null, 0.0, null, 1.0, null, null, null, 8, null, "T", null, "under 1.2% mapping rate", "illumina", "nextseq", "unknown", "random_priming", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-07-07", "Undetermined", "Adult", "Kidney", "Renal System"], [42605, "SRR5810680", "SRX2989236", "SRS2341159", "SRP111339", "PRJNA393429", "Dissecting hematopoietic and renal cell heterogeneity in adult zebrafish at single cell resolution using RNA sequencing [inDrops]", "GSE100910", "Transcriptome Analysis", "Recent advances in single cell transcriptomic profiling have provided unprecedented access to investigate cell heterogeneity during tissue and organ development. Here we utilized massively parallel single cell RNA sequencing to define cell heterogeneity within the zebrafish kidney marrow constructing a comprehensive molecular atlas of definitive hematopoiesis and functionally distinct renal cells found in adult zebrafish. Because our method analyzed blood and kidney cells in an unbiased manner our approach was useful in characterizing immune cell deficiencies within prkdcD3612fs il2rgaY91fs and double homozygous mutant fish identifying blood cell losses in T B and natural killer cells within specific genetic mutants. Our analysis also uncovered novel cell types including two classes of natural killer immune cells classically defined and erythroid primed hematopoietic stem and progenitor cells mucin secreting kidney cells and kidney stem/progenitor cells. In total our work provides the first comprehensive single cell transcriptomic analysis of kidney and marrow cells in the adult zebrafish. Overall design: The goal of our study is to establish the transcriptional profiles of hematopoietic and kidney cell lineages residing in the zebrafish whole kidney marrow. Firstly we performed single cell RNA sequencing by a modified Smart seq2 protocol on sorted single cells from fluorescent transgenic zebrafish lines which label distinct blood cell types n = 246 cells total. Secondly we utilized droplet based single cell RNA sequencing inDrop to investigate unmarked comprehensive hematopoietic lineage structure within wild type casper strain zebrafish N=3 animals n=3 782 cells total. From this we identified ten distinct hematopoietic groups of blood and immune identities. Thirdly we confirmed blood lineage interpretations by comparing hematopoietic lineages within wild type fish with mutant zebrafish with known immunodeficiencies including prkdcD3612fs N=3 animals n=3 201 cells il2rgaY91fs N=2 animals n=2 068 cells and prkdcD3612fs il2rgaY91fs double compound mutant fish N=2 animals n=2 276 cells. Lastly we identified seven structural and functional cell lineages of kidney identities in the whole kidney marrow n=1 699 kidney cells.", "parent bioproject:PRJNA393415", "pubmed:28878000", null, "inDrop sequencing of multiple samples 1", "GSM2696103", null, "source name:Whole kidney marrow|indrop version:inDrop V3 protocol|tissue:kidney marrow|genotype:multiple genotypes", "inDrop sequencing of multiple samples 1", "Reads were processed using the indrops pipelinehttps://github.com/indrops/indrops. Briefly reads were filtered according to structure and quality criteria. Filtered reads were then they were sorted by barcode. Demultiplexed reads were aligned to the GRCz10 transcriptome using Bowtie. Please see file \"inDrop GEO supplementary.xlsx \" available on the series record for information on de multiplexing and barcodes for individual samples and cells. Genome build: GRCz10 Supplementary files format and content: text file with read counts.", "Whole kidney marrow", null, "RNA from individual cells were reverse transcribed barcoded and unique transcripts are imprinted with UMI all within the droplet. RNA from 1 500 cells of each animal were pooled and subsequently processed together. cDNA products were pre amplified to produce a cDNA library compatible with the Illumina Nextseq platform. Three sequencing runs were completed using the Nextseq 500 High Output V2 kit 75 cycles Illumina on a NextSeq 500 platform Illumina. Zebrafish cells are isolated from the kidney marrow into single cell suspensions and treated with 15% Optiprep Sigma. Single cells are microfluidically sorted into droplets containing reverse transcription and barcoding reagents. This method is outlined in Klein et al. PMID 26000487 and Zilionis et al. PMID 27929523.", null, "indrop version:inDrop V3 protocol|tissue:kidney marrow|genotype:multiple genotypes", "GSM2696103", "GSM2696103: inDrop sequencing of multiple samples 1; Danio rerio; RNA Seq", "GSM2696103", null, "1", "RNA from individual cells were reverse transcribed barcoded and unique transcripts are imprinted with UMI all within the droplet. RNA from 1 500 cells of each animal were pooled and subsequently processed together. cDNA products were pre amplified to produce a cDNA library compatible with the Illumina Nextseq platform. Three sequencing runs were completed using the Nextseq 500 High Output V2 kit 75 cycles Illumina on a NextSeq 500 platform Illumina. Zebrafish cells are isolated from the kidney marrow into single cell suspensions and treated with 15% Optiprep Sigma. Single cells are microfluidically sorted into droplets containing reverse transcription and barcoding reagents. This method is outlined in Klein et al. PMID 26000487 and Zilionis et al. PMID 27929523.", "GEO Accession:GSM2696103", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP111339", null, null, "inDrop_v3_run1_R3.fastq.gz", "fastq", 3523720792.0, 440465099.0, "GSM2696103 r3", "0:8", "A:1016854893;C:682256486;G:759860179;T:1063129659;N:1619575", 8, null, null, null, 1016854893, 682256486, 759860179, 1063129659, 1619575, "SRX2989236", "SRS2341159", "SRA584587", "GEO", "Pathology, Massachusetts General Hospital", 1, 0.0, null, 0.0, null, 1.0, null, null, null, 8, null, "T", null, "under 1.2% mapping rate", "illumina", "nextseq", "unknown", "random_priming", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-07-07", "Undetermined", "Adult", "Kidney", "Renal System"], [42606, "SRR5810681", "SRX2989236", "SRS2341159", "SRP111339", "PRJNA393429", "Dissecting hematopoietic and renal cell heterogeneity in adult zebrafish at single cell resolution using RNA sequencing [inDrops]", "GSE100910", "Transcriptome Analysis", "Recent advances in single cell transcriptomic profiling have provided unprecedented access to investigate cell heterogeneity during tissue and organ development. Here we utilized massively parallel single cell RNA sequencing to define cell heterogeneity within the zebrafish kidney marrow constructing a comprehensive molecular atlas of definitive hematopoiesis and functionally distinct renal cells found in adult zebrafish. Because our method analyzed blood and kidney cells in an unbiased manner our approach was useful in characterizing immune cell deficiencies within prkdcD3612fs il2rgaY91fs and double homozygous mutant fish identifying blood cell losses in T B and natural killer cells within specific genetic mutants. Our analysis also uncovered novel cell types including two classes of natural killer immune cells classically defined and erythroid primed hematopoietic stem and progenitor cells mucin secreting kidney cells and kidney stem/progenitor cells. In total our work provides the first comprehensive single cell transcriptomic analysis of kidney and marrow cells in the adult zebrafish. Overall design: The goal of our study is to establish the transcriptional profiles of hematopoietic and kidney cell lineages residing in the zebrafish whole kidney marrow. Firstly we performed single cell RNA sequencing by a modified Smart seq2 protocol on sorted single cells from fluorescent transgenic zebrafish lines which label distinct blood cell types n = 246 cells total. Secondly we utilized droplet based single cell RNA sequencing inDrop to investigate unmarked comprehensive hematopoietic lineage structure within wild type casper strain zebrafish N=3 animals n=3 782 cells total. From this we identified ten distinct hematopoietic groups of blood and immune identities. Thirdly we confirmed blood lineage interpretations by comparing hematopoietic lineages within wild type fish with mutant zebrafish with known immunodeficiencies including prkdcD3612fs N=3 animals n=3 201 cells il2rgaY91fs N=2 animals n=2 068 cells and prkdcD3612fs il2rgaY91fs double compound mutant fish N=2 animals n=2 276 cells. Lastly we identified seven structural and functional cell lineages of kidney identities in the whole kidney marrow n=1 699 kidney cells.", "parent bioproject:PRJNA393415", "pubmed:28878000", null, "inDrop sequencing of multiple samples 1", "GSM2696103", null, "source name:Whole kidney marrow|indrop version:inDrop V3 protocol|tissue:kidney marrow|genotype:multiple genotypes", "inDrop sequencing of multiple samples 1", "Reads were processed using the indrops pipelinehttps://github.com/indrops/indrops. Briefly reads were filtered according to structure and quality criteria. Filtered reads were then they were sorted by barcode. Demultiplexed reads were aligned to the GRCz10 transcriptome using Bowtie. Please see file \"inDrop GEO supplementary.xlsx \" available on the series record for information on de multiplexing and barcodes for individual samples and cells. Genome build: GRCz10 Supplementary files format and content: text file with read counts.", "Whole kidney marrow", null, "RNA from individual cells were reverse transcribed barcoded and unique transcripts are imprinted with UMI all within the droplet. RNA from 1 500 cells of each animal were pooled and subsequently processed together. cDNA products were pre amplified to produce a cDNA library compatible with the Illumina Nextseq platform. Three sequencing runs were completed using the Nextseq 500 High Output V2 kit 75 cycles Illumina on a NextSeq 500 platform Illumina. Zebrafish cells are isolated from the kidney marrow into single cell suspensions and treated with 15% Optiprep Sigma. Single cells are microfluidically sorted into droplets containing reverse transcription and barcoding reagents. This method is outlined in Klein et al. PMID 26000487 and Zilionis et al. PMID 27929523.", null, "indrop version:inDrop V3 protocol|tissue:kidney marrow|genotype:multiple genotypes", "GSM2696103", "GSM2696103: inDrop sequencing of multiple samples 1; Danio rerio; RNA Seq", "GSM2696103", null, "1", "RNA from individual cells were reverse transcribed barcoded and unique transcripts are imprinted with UMI all within the droplet. RNA from 1 500 cells of each animal were pooled and subsequently processed together. cDNA products were pre amplified to produce a cDNA library compatible with the Illumina Nextseq platform. Three sequencing runs were completed using the Nextseq 500 High Output V2 kit 75 cycles Illumina on a NextSeq 500 platform Illumina. Zebrafish cells are isolated from the kidney marrow into single cell suspensions and treated with 15% Optiprep Sigma. Single cells are microfluidically sorted into droplets containing reverse transcription and barcoding reagents. This method is outlined in Klein et al. PMID 26000487 and Zilionis et al. PMID 27929523.", "GEO Accession:GSM2696103", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP111339", null, null, "inDrop_v3_run1_R4.fastq.gz", "fastq", 6166511386.0, 440465099.0, "GSM2696103 r4", "0:14", "A:1513770161;C:1469643118;G:1669791951;T:1509862301;N:3443855", 14, null, null, null, 1513770161, 1469643118, 1669791951, 1509862301, 3443855, "SRX2989236", "SRS2341159", "SRA584587", "GEO", "Pathology, Massachusetts General Hospital", 1, 0.0, null, 0.0, null, 1.0, null, null, null, 14, null, "T", null, "under 1.2% mapping rate", "illumina", "nextseq", "unknown", "random_priming", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-07-07", "Undetermined", "Adult", "Kidney", "Renal System"], [42607, "SRR5810676", "SRX2989235", "SRS2341158", "SRP111339", "PRJNA393429", "Dissecting hematopoietic and renal cell heterogeneity in adult zebrafish at single cell resolution using RNA sequencing [inDrops]", "GSE100910", "Transcriptome Analysis", "Recent advances in single cell transcriptomic profiling have provided unprecedented access to investigate cell heterogeneity during tissue and organ development. Here we utilized massively parallel single cell RNA sequencing to define cell heterogeneity within the zebrafish kidney marrow constructing a comprehensive molecular atlas of definitive hematopoiesis and functionally distinct renal cells found in adult zebrafish. Because our method analyzed blood and kidney cells in an unbiased manner our approach was useful in characterizing immune cell deficiencies within prkdcD3612fs il2rgaY91fs and double homozygous mutant fish identifying blood cell losses in T B and natural killer cells within specific genetic mutants. Our analysis also uncovered novel cell types including two classes of natural killer immune cells classically defined and erythroid primed hematopoietic stem and progenitor cells mucin secreting kidney cells and kidney stem/progenitor cells. In total our work provides the first comprehensive single cell transcriptomic analysis of kidney and marrow cells in the adult zebrafish. Overall design: The goal of our study is to establish the transcriptional profiles of hematopoietic and kidney cell lineages residing in the zebrafish whole kidney marrow. Firstly we performed single cell RNA sequencing by a modified Smart seq2 protocol on sorted single cells from fluorescent transgenic zebrafish lines which label distinct blood cell types n = 246 cells total. Secondly we utilized droplet based single cell RNA sequencing inDrop to investigate unmarked comprehensive hematopoietic lineage structure within wild type casper strain zebrafish N=3 animals n=3 782 cells total. From this we identified ten distinct hematopoietic groups of blood and immune identities. Thirdly we confirmed blood lineage interpretations by comparing hematopoietic lineages within wild type fish with mutant zebrafish with known immunodeficiencies including prkdcD3612fs N=3 animals n=3 201 cells il2rgaY91fs N=2 animals n=2 068 cells and prkdcD3612fs il2rgaY91fs double compound mutant fish N=2 animals n=2 276 cells. Lastly we identified seven structural and functional cell lineages of kidney identities in the whole kidney marrow n=1 699 kidney cells.", "parent bioproject:PRJNA393415", "pubmed:28878000", null, "inDrop sequencing of prkdcD3612fs homozygous mutant zebrafish sample animal #2", "GSM2696102", null, "source name:Whole kidney marrow|indrop version:inDrop V2 protocol|tissue:kidney marrow|genotype:prkdcD3612fs", "inDrop sequencing of prkdcD3612fs homozygous mutant zebrafish sample animal #2", "Reads were processed using the indrops pipelinehttps://github.com/indrops/indrops. Briefly reads were filtered according to structure and quality criteria. Filtered reads were then they were sorted by barcode. Demultiplexed reads were aligned to the GRCz10 transcriptome using Bowtie. Please see file \"inDrop GEO supplementary.xlsx \" available on the series record for information on de multiplexing and barcodes for individual samples and cells. Genome build: GRCz10 Supplementary files format and content: text file with read counts.", "Whole kidney marrow", null, "RNA from individual cells were reverse transcribed barcoded and unique transcripts are imprinted with UMI all within the droplet. RNA from 1 500 cells of each animal were pooled and subsequently processed together. cDNA products were pre amplified to produce a cDNA library compatible with the Illumina Nextseq platform. Three sequencing runs were completed using the Nextseq 500 High Output V2 kit 75 cycles Illumina on a NextSeq 500 platform Illumina. Zebrafish cells are isolated from the kidney marrow into single cell suspensions and treated with 15% Optiprep Sigma. Single cells are microfluidically sorted into droplets containing reverse transcription and barcoding reagents. This method is outlined in Klein et al. PMID 26000487 and Zilionis et al. PMID 27929523.", null, "indrop version:inDrop V2 protocol|tissue:kidney marrow|genotype:prkdcD3612fs", "GSM2696102", "GSM2696102: inDrop sequencing of prkdcD3612fs homozygous mutant zebrafish sample animal #2; Danio rerio; RNA Seq", "GSM2696102", null, "1", "RNA from individual cells were reverse transcribed barcoded and unique transcripts are imprinted with UMI all within the droplet. RNA from 1 500 cells of each animal were pooled and subsequently processed together. cDNA products were pre amplified to produce a cDNA library compatible with the Illumina Nextseq platform. Three sequencing runs were completed using the Nextseq 500 High Output V2 kit 75 cycles Illumina on a NextSeq 500 platform Illumina. Zebrafish cells are isolated from the kidney marrow into single cell suspensions and treated with 15% Optiprep Sigma. Single cells are microfluidically sorted into droplets containing reverse transcription and barcoding reagents. This method is outlined in Klein et al. PMID 26000487 and Zilionis et al. PMID 27929523.", "GEO Accession:GSM2696102", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP111339", null, null, "PRKDC2_R1.fastq.gz", "fastq", 1987929396.0, 55220261.0, "GSM2696102 r1", "0:36 1:0", "A:506372087;C:413396954;G:415448050;T:652683387;N:28918", 36, 0, null, null, 506372087, 413396954, 415448050, 652683387, 28918, "SRX2989235", "SRS2341158", "SRA584587", "GEO", "Pathology, Massachusetts General Hospital", 1, 0.86587, null, 0.12447, null, 0.80336, null, 0.54126, null, 36, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "random_priming", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-07-07", "Undetermined", "Adult", "Kidney", "Renal System"], [42608, "SRR5810677", "SRX2989235", "SRS2341158", "SRP111339", "PRJNA393429", "Dissecting hematopoietic and renal cell heterogeneity in adult zebrafish at single cell resolution using RNA sequencing [inDrops]", "GSE100910", "Transcriptome Analysis", "Recent advances in single cell transcriptomic profiling have provided unprecedented access to investigate cell heterogeneity during tissue and organ development. Here we utilized massively parallel single cell RNA sequencing to define cell heterogeneity within the zebrafish kidney marrow constructing a comprehensive molecular atlas of definitive hematopoiesis and functionally distinct renal cells found in adult zebrafish. Because our method analyzed blood and kidney cells in an unbiased manner our approach was useful in characterizing immune cell deficiencies within prkdcD3612fs il2rgaY91fs and double homozygous mutant fish identifying blood cell losses in T B and natural killer cells within specific genetic mutants. Our analysis also uncovered novel cell types including two classes of natural killer immune cells classically defined and erythroid primed hematopoietic stem and progenitor cells mucin secreting kidney cells and kidney stem/progenitor cells. In total our work provides the first comprehensive single cell transcriptomic analysis of kidney and marrow cells in the adult zebrafish. Overall design: The goal of our study is to establish the transcriptional profiles of hematopoietic and kidney cell lineages residing in the zebrafish whole kidney marrow. Firstly we performed single cell RNA sequencing by a modified Smart seq2 protocol on sorted single cells from fluorescent transgenic zebrafish lines which label distinct blood cell types n = 246 cells total. Secondly we utilized droplet based single cell RNA sequencing inDrop to investigate unmarked comprehensive hematopoietic lineage structure within wild type casper strain zebrafish N=3 animals n=3 782 cells total. From this we identified ten distinct hematopoietic groups of blood and immune identities. Thirdly we confirmed blood lineage interpretations by comparing hematopoietic lineages within wild type fish with mutant zebrafish with known immunodeficiencies including prkdcD3612fs N=3 animals n=3 201 cells il2rgaY91fs N=2 animals n=2 068 cells and prkdcD3612fs il2rgaY91fs double compound mutant fish N=2 animals n=2 276 cells. Lastly we identified seven structural and functional cell lineages of kidney identities in the whole kidney marrow n=1 699 kidney cells.", "parent bioproject:PRJNA393415", "pubmed:28878000", null, "inDrop sequencing of prkdcD3612fs homozygous mutant zebrafish sample animal #2", "GSM2696102", null, "source name:Whole kidney marrow|indrop version:inDrop V2 protocol|tissue:kidney marrow|genotype:prkdcD3612fs", "inDrop sequencing of prkdcD3612fs homozygous mutant zebrafish sample animal #2", "Reads were processed using the indrops pipelinehttps://github.com/indrops/indrops. Briefly reads were filtered according to structure and quality criteria. Filtered reads were then they were sorted by barcode. Demultiplexed reads were aligned to the GRCz10 transcriptome using Bowtie. Please see file \"inDrop GEO supplementary.xlsx \" available on the series record for information on de multiplexing and barcodes for individual samples and cells. Genome build: GRCz10 Supplementary files format and content: text file with read counts.", "Whole kidney marrow", null, "RNA from individual cells were reverse transcribed barcoded and unique transcripts are imprinted with UMI all within the droplet. RNA from 1 500 cells of each animal were pooled and subsequently processed together. cDNA products were pre amplified to produce a cDNA library compatible with the Illumina Nextseq platform. Three sequencing runs were completed using the Nextseq 500 High Output V2 kit 75 cycles Illumina on a NextSeq 500 platform Illumina. Zebrafish cells are isolated from the kidney marrow into single cell suspensions and treated with 15% Optiprep Sigma. Single cells are microfluidically sorted into droplets containing reverse transcription and barcoding reagents. This method is outlined in Klein et al. PMID 26000487 and Zilionis et al. PMID 27929523.", null, "indrop version:inDrop V2 protocol|tissue:kidney marrow|genotype:prkdcD3612fs", "GSM2696102", "GSM2696102: inDrop sequencing of prkdcD3612fs homozygous mutant zebrafish sample animal #2; Danio rerio; RNA Seq", "GSM2696102", null, "1", "RNA from individual cells were reverse transcribed barcoded and unique transcripts are imprinted with UMI all within the droplet. RNA from 1 500 cells of each animal were pooled and subsequently processed together. cDNA products were pre amplified to produce a cDNA library compatible with the Illumina Nextseq platform. Three sequencing runs were completed using the Nextseq 500 High Output V2 kit 75 cycles Illumina on a NextSeq 500 platform Illumina. Zebrafish cells are isolated from the kidney marrow into single cell suspensions and treated with 15% Optiprep Sigma. Single cells are microfluidically sorted into droplets containing reverse transcription and barcoding reagents. This method is outlined in Klein et al. PMID 26000487 and Zilionis et al. PMID 27929523.", "GEO Accession:GSM2696102", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP111339", null, null, "PRKDC2_R2.fastq.gz", "fastq", 2761013050.0, 55220261.0, "GSM2696102 r2", "0:0 1:50", "A:554483675;C:506568509;G:721419849;T:974751566;N:3789451", 0, 50, null, null, 554483675, 506568509, 721419849, 974751566, 3789451, "SRX2989235", "SRS2341158", "SRA584587", "GEO", "Pathology, Massachusetts General Hospital", 1, 0.00796, null, 0.0077, null, 0.99961, null, 0.78947, null, 50, null, "T", null, "under 1.2% mapping rate", "illumina", "nextseq", "unknown", "random_priming", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-07-07", "Undetermined", "Adult", "Kidney", "Renal System"], [42609, "SRR5810674", "SRX2989234", "SRS2341157", "SRP111339", "PRJNA393429", "Dissecting hematopoietic and renal cell heterogeneity in adult zebrafish at single cell resolution using RNA sequencing [inDrops]", "GSE100910", "Transcriptome Analysis", "Recent advances in single cell transcriptomic profiling have provided unprecedented access to investigate cell heterogeneity during tissue and organ development. Here we utilized massively parallel single cell RNA sequencing to define cell heterogeneity within the zebrafish kidney marrow constructing a comprehensive molecular atlas of definitive hematopoiesis and functionally distinct renal cells found in adult zebrafish. Because our method analyzed blood and kidney cells in an unbiased manner our approach was useful in characterizing immune cell deficiencies within prkdcD3612fs il2rgaY91fs and double homozygous mutant fish identifying blood cell losses in T B and natural killer cells within specific genetic mutants. Our analysis also uncovered novel cell types including two classes of natural killer immune cells classically defined and erythroid primed hematopoietic stem and progenitor cells mucin secreting kidney cells and kidney stem/progenitor cells. In total our work provides the first comprehensive single cell transcriptomic analysis of kidney and marrow cells in the adult zebrafish. Overall design: The goal of our study is to establish the transcriptional profiles of hematopoietic and kidney cell lineages residing in the zebrafish whole kidney marrow. Firstly we performed single cell RNA sequencing by a modified Smart seq2 protocol on sorted single cells from fluorescent transgenic zebrafish lines which label distinct blood cell types n = 246 cells total. Secondly we utilized droplet based single cell RNA sequencing inDrop to investigate unmarked comprehensive hematopoietic lineage structure within wild type casper strain zebrafish N=3 animals n=3 782 cells total. From this we identified ten distinct hematopoietic groups of blood and immune identities. Thirdly we confirmed blood lineage interpretations by comparing hematopoietic lineages within wild type fish with mutant zebrafish with known immunodeficiencies including prkdcD3612fs N=3 animals n=3 201 cells il2rgaY91fs N=2 animals n=2 068 cells and prkdcD3612fs il2rgaY91fs double compound mutant fish N=2 animals n=2 276 cells. Lastly we identified seven structural and functional cell lineages of kidney identities in the whole kidney marrow n=1 699 kidney cells.", "parent bioproject:PRJNA393415", "pubmed:28878000", null, "inDrop sequencing of prkdcD3612fs homozygous mutant zebrafish sample animal #1", "GSM2696101", null, "source name:Whole kidney marrow|indrop version:inDrop V2 protocol|tissue:kidney marrow|genotype:prkdcD3612fs", "inDrop sequencing of prkdcD3612fs homozygous mutant zebrafish sample animal #1", "Reads were processed using the indrops pipelinehttps://github.com/indrops/indrops. Briefly reads were filtered according to structure and quality criteria. Filtered reads were then they were sorted by barcode. Demultiplexed reads were aligned to the GRCz10 transcriptome using Bowtie. Please see file \"inDrop GEO supplementary.xlsx \" available on the series record for information on de multiplexing and barcodes for individual samples and cells. Genome build: GRCz10 Supplementary files format and content: text file with read counts.", "Whole kidney marrow", null, "RNA from individual cells were reverse transcribed barcoded and unique transcripts are imprinted with UMI all within the droplet. RNA from 1 500 cells of each animal were pooled and subsequently processed together. cDNA products were pre amplified to produce a cDNA library compatible with the Illumina Nextseq platform. Three sequencing runs were completed using the Nextseq 500 High Output V2 kit 75 cycles Illumina on a NextSeq 500 platform Illumina. Zebrafish cells are isolated from the kidney marrow into single cell suspensions and treated with 15% Optiprep Sigma. Single cells are microfluidically sorted into droplets containing reverse transcription and barcoding reagents. This method is outlined in Klein et al. PMID 26000487 and Zilionis et al. PMID 27929523.", null, "indrop version:inDrop V2 protocol|tissue:kidney marrow|genotype:prkdcD3612fs", "GSM2696101", "GSM2696101: inDrop sequencing of prkdcD3612fs homozygous mutant zebrafish sample animal #1; Danio rerio; RNA Seq", "GSM2696101", null, "1", "RNA from individual cells were reverse transcribed barcoded and unique transcripts are imprinted with UMI all within the droplet. RNA from 1 500 cells of each animal were pooled and subsequently processed together. cDNA products were pre amplified to produce a cDNA library compatible with the Illumina Nextseq platform. Three sequencing runs were completed using the Nextseq 500 High Output V2 kit 75 cycles Illumina on a NextSeq 500 platform Illumina. Zebrafish cells are isolated from the kidney marrow into single cell suspensions and treated with 15% Optiprep Sigma. Single cells are microfluidically sorted into droplets containing reverse transcription and barcoding reagents. This method is outlined in Klein et al. PMID 26000487 and Zilionis et al. PMID 27929523.", "GEO Accession:GSM2696101", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP111339", null, null, "PRKDC1_R1.fastq.gz", "fastq", 1549442736.0, 43040076.0, "GSM2696101 r1", "0:36 1:0", "A:402261661;C:326108405;G:319637981;T:501413963;N:20726", 36, 0, null, null, 402261661, 326108405, 319637981, 501413963, 20726, "SRX2989234", "SRS2341157", "SRA584587", "GEO", "Pathology, Massachusetts General Hospital", 1, 0.84263, null, 0.11449, null, 0.79464, null, 0.57001, null, 36, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "random_priming", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-07-07", "Undetermined", "Adult", "Kidney", "Renal System"], [42610, "SRR5810675", "SRX2989234", "SRS2341157", "SRP111339", "PRJNA393429", "Dissecting hematopoietic and renal cell heterogeneity in adult zebrafish at single cell resolution using RNA sequencing [inDrops]", "GSE100910", "Transcriptome Analysis", "Recent advances in single cell transcriptomic profiling have provided unprecedented access to investigate cell heterogeneity during tissue and organ development. Here we utilized massively parallel single cell RNA sequencing to define cell heterogeneity within the zebrafish kidney marrow constructing a comprehensive molecular atlas of definitive hematopoiesis and functionally distinct renal cells found in adult zebrafish. Because our method analyzed blood and kidney cells in an unbiased manner our approach was useful in characterizing immune cell deficiencies within prkdcD3612fs il2rgaY91fs and double homozygous mutant fish identifying blood cell losses in T B and natural killer cells within specific genetic mutants. Our analysis also uncovered novel cell types including two classes of natural killer immune cells classically defined and erythroid primed hematopoietic stem and progenitor cells mucin secreting kidney cells and kidney stem/progenitor cells. In total our work provides the first comprehensive single cell transcriptomic analysis of kidney and marrow cells in the adult zebrafish. Overall design: The goal of our study is to establish the transcriptional profiles of hematopoietic and kidney cell lineages residing in the zebrafish whole kidney marrow. Firstly we performed single cell RNA sequencing by a modified Smart seq2 protocol on sorted single cells from fluorescent transgenic zebrafish lines which label distinct blood cell types n = 246 cells total. Secondly we utilized droplet based single cell RNA sequencing inDrop to investigate unmarked comprehensive hematopoietic lineage structure within wild type casper strain zebrafish N=3 animals n=3 782 cells total. From this we identified ten distinct hematopoietic groups of blood and immune identities. Thirdly we confirmed blood lineage interpretations by comparing hematopoietic lineages within wild type fish with mutant zebrafish with known immunodeficiencies including prkdcD3612fs N=3 animals n=3 201 cells il2rgaY91fs N=2 animals n=2 068 cells and prkdcD3612fs il2rgaY91fs double compound mutant fish N=2 animals n=2 276 cells. Lastly we identified seven structural and functional cell lineages of kidney identities in the whole kidney marrow n=1 699 kidney cells.", "parent bioproject:PRJNA393415", "pubmed:28878000", null, "inDrop sequencing of prkdcD3612fs homozygous mutant zebrafish sample animal #1", "GSM2696101", null, "source name:Whole kidney marrow|indrop version:inDrop V2 protocol|tissue:kidney marrow|genotype:prkdcD3612fs", "inDrop sequencing of prkdcD3612fs homozygous mutant zebrafish sample animal #1", "Reads were processed using the indrops pipelinehttps://github.com/indrops/indrops. Briefly reads were filtered according to structure and quality criteria. Filtered reads were then they were sorted by barcode. Demultiplexed reads were aligned to the GRCz10 transcriptome using Bowtie. Please see file \"inDrop GEO supplementary.xlsx \" available on the series record for information on de multiplexing and barcodes for individual samples and cells. Genome build: GRCz10 Supplementary files format and content: text file with read counts.", "Whole kidney marrow", null, "RNA from individual cells were reverse transcribed barcoded and unique transcripts are imprinted with UMI all within the droplet. RNA from 1 500 cells of each animal were pooled and subsequently processed together. cDNA products were pre amplified to produce a cDNA library compatible with the Illumina Nextseq platform. Three sequencing runs were completed using the Nextseq 500 High Output V2 kit 75 cycles Illumina on a NextSeq 500 platform Illumina. Zebrafish cells are isolated from the kidney marrow into single cell suspensions and treated with 15% Optiprep Sigma. Single cells are microfluidically sorted into droplets containing reverse transcription and barcoding reagents. This method is outlined in Klein et al. PMID 26000487 and Zilionis et al. PMID 27929523.", null, "indrop version:inDrop V2 protocol|tissue:kidney marrow|genotype:prkdcD3612fs", "GSM2696101", "GSM2696101: inDrop sequencing of prkdcD3612fs homozygous mutant zebrafish sample animal #1; Danio rerio; RNA Seq", "GSM2696101", null, "1", "RNA from individual cells were reverse transcribed barcoded and unique transcripts are imprinted with UMI all within the droplet. RNA from 1 500 cells of each animal were pooled and subsequently processed together. cDNA products were pre amplified to produce a cDNA library compatible with the Illumina Nextseq platform. Three sequencing runs were completed using the Nextseq 500 High Output V2 kit 75 cycles Illumina on a NextSeq 500 platform Illumina. Zebrafish cells are isolated from the kidney marrow into single cell suspensions and treated with 15% Optiprep Sigma. Single cells are microfluidically sorted into droplets containing reverse transcription and barcoding reagents. This method is outlined in Klein et al. PMID 26000487 and Zilionis et al. PMID 27929523.", "GEO Accession:GSM2696101", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP111339", null, null, "PRKDC1_R2.fastq.gz", "fastq", 2152003800.0, 43040076.0, "GSM2696101 r2", "0:0 1:50", "A:432547886;C:403759182;G:561234660;T:751564536;N:2897536", 0, 50, null, null, 432547886, 403759182, 561234660, 751564536, 2897536, "SRX2989234", "SRS2341157", "SRA584587", "GEO", "Pathology, Massachusetts General Hospital", 1, 0.00796, null, 0.00773, null, 0.99971, null, 0.82758, null, 50, null, "T", null, "under 1.2% mapping rate", "illumina", "nextseq", "unknown", "random_priming", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-07-07", "Undetermined", "Adult", "Kidney", "Renal System"], [42611, "SRR5810672", "SRX2989233", "SRS2341156", "SRP111339", "PRJNA393429", "Dissecting hematopoietic and renal cell heterogeneity in adult zebrafish at single cell resolution using RNA sequencing [inDrops]", "GSE100910", "Transcriptome Analysis", "Recent advances in single cell transcriptomic profiling have provided unprecedented access to investigate cell heterogeneity during tissue and organ development. Here we utilized massively parallel single cell RNA sequencing to define cell heterogeneity within the zebrafish kidney marrow constructing a comprehensive molecular atlas of definitive hematopoiesis and functionally distinct renal cells found in adult zebrafish. Because our method analyzed blood and kidney cells in an unbiased manner our approach was useful in characterizing immune cell deficiencies within prkdcD3612fs il2rgaY91fs and double homozygous mutant fish identifying blood cell losses in T B and natural killer cells within specific genetic mutants. Our analysis also uncovered novel cell types including two classes of natural killer immune cells classically defined and erythroid primed hematopoietic stem and progenitor cells mucin secreting kidney cells and kidney stem/progenitor cells. In total our work provides the first comprehensive single cell transcriptomic analysis of kidney and marrow cells in the adult zebrafish. Overall design: The goal of our study is to establish the transcriptional profiles of hematopoietic and kidney cell lineages residing in the zebrafish whole kidney marrow. Firstly we performed single cell RNA sequencing by a modified Smart seq2 protocol on sorted single cells from fluorescent transgenic zebrafish lines which label distinct blood cell types n = 246 cells total. Secondly we utilized droplet based single cell RNA sequencing inDrop to investigate unmarked comprehensive hematopoietic lineage structure within wild type casper strain zebrafish N=3 animals n=3 782 cells total. From this we identified ten distinct hematopoietic groups of blood and immune identities. Thirdly we confirmed blood lineage interpretations by comparing hematopoietic lineages within wild type fish with mutant zebrafish with known immunodeficiencies including prkdcD3612fs N=3 animals n=3 201 cells il2rgaY91fs N=2 animals n=2 068 cells and prkdcD3612fs il2rgaY91fs double compound mutant fish N=2 animals n=2 276 cells. Lastly we identified seven structural and functional cell lineages of kidney identities in the whole kidney marrow n=1 699 kidney cells.", "parent bioproject:PRJNA393415", "pubmed:28878000", null, "inDrop sequencing of WT sample animal #2", "GSM2696100", null, "source name:Whole kidney marrow|indrop version:inDrop V2 protocol|tissue:kidney marrow|genotype:wildtype", "inDrop sequencing of WT sample animal #2", "Reads were processed using the indrops pipelinehttps://github.com/indrops/indrops. Briefly reads were filtered according to structure and quality criteria. Filtered reads were then they were sorted by barcode. Demultiplexed reads were aligned to the GRCz10 transcriptome using Bowtie. Please see file \"inDrop GEO supplementary.xlsx \" available on the series record for information on de multiplexing and barcodes for individual samples and cells. Genome build: GRCz10 Supplementary files format and content: text file with read counts.", "Whole kidney marrow", null, "RNA from individual cells were reverse transcribed barcoded and unique transcripts are imprinted with UMI all within the droplet. RNA from 1 500 cells of each animal were pooled and subsequently processed together. cDNA products were pre amplified to produce a cDNA library compatible with the Illumina Nextseq platform. Three sequencing runs were completed using the Nextseq 500 High Output V2 kit 75 cycles Illumina on a NextSeq 500 platform Illumina. Zebrafish cells are isolated from the kidney marrow into single cell suspensions and treated with 15% Optiprep Sigma. Single cells are microfluidically sorted into droplets containing reverse transcription and barcoding reagents. This method is outlined in Klein et al. PMID 26000487 and Zilionis et al. PMID 27929523.", null, "indrop version:inDrop V2 protocol|tissue:kidney marrow|genotype:wildtype", "GSM2696100", "GSM2696100: inDrop sequencing of WT sample animal #2; Danio rerio; RNA Seq", "GSM2696100", null, "1", "RNA from individual cells were reverse transcribed barcoded and unique transcripts are imprinted with UMI all within the droplet. RNA from 1 500 cells of each animal were pooled and subsequently processed together. cDNA products were pre amplified to produce a cDNA library compatible with the Illumina Nextseq platform. Three sequencing runs were completed using the Nextseq 500 High Output V2 kit 75 cycles Illumina on a NextSeq 500 platform Illumina. Zebrafish cells are isolated from the kidney marrow into single cell suspensions and treated with 15% Optiprep Sigma. Single cells are microfluidically sorted into droplets containing reverse transcription and barcoding reagents. This method is outlined in Klein et al. PMID 26000487 and Zilionis et al. PMID 27929523.", "GEO Accession:GSM2696100", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP111339", null, null, "WT2_R1.fastq.gz", "fastq", 3202042500.0, 88945625.0, "GSM2696100 r1", "0:36 1:0", "A:821476951;C:657776656;G:665293293;T:1057448635;N:46965", 36, 0, null, null, 821476951, 657776656, 665293293, 1057448635, 46965, "SRX2989233", "SRS2341156", "SRA584587", "GEO", "Pathology, Massachusetts General Hospital", 1, 0.84222, null, 0.12969, null, 0.80683, null, 0.53542, null, 36, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "random_priming", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-07-07", "Undetermined", "Adult", "Kidney", "Renal System"], [42612, "SRR5810673", "SRX2989233", "SRS2341156", "SRP111339", "PRJNA393429", "Dissecting hematopoietic and renal cell heterogeneity in adult zebrafish at single cell resolution using RNA sequencing [inDrops]", "GSE100910", "Transcriptome Analysis", "Recent advances in single cell transcriptomic profiling have provided unprecedented access to investigate cell heterogeneity during tissue and organ development. Here we utilized massively parallel single cell RNA sequencing to define cell heterogeneity within the zebrafish kidney marrow constructing a comprehensive molecular atlas of definitive hematopoiesis and functionally distinct renal cells found in adult zebrafish. Because our method analyzed blood and kidney cells in an unbiased manner our approach was useful in characterizing immune cell deficiencies within prkdcD3612fs il2rgaY91fs and double homozygous mutant fish identifying blood cell losses in T B and natural killer cells within specific genetic mutants. Our analysis also uncovered novel cell types including two classes of natural killer immune cells classically defined and erythroid primed hematopoietic stem and progenitor cells mucin secreting kidney cells and kidney stem/progenitor cells. In total our work provides the first comprehensive single cell transcriptomic analysis of kidney and marrow cells in the adult zebrafish. Overall design: The goal of our study is to establish the transcriptional profiles of hematopoietic and kidney cell lineages residing in the zebrafish whole kidney marrow. Firstly we performed single cell RNA sequencing by a modified Smart seq2 protocol on sorted single cells from fluorescent transgenic zebrafish lines which label distinct blood cell types n = 246 cells total. Secondly we utilized droplet based single cell RNA sequencing inDrop to investigate unmarked comprehensive hematopoietic lineage structure within wild type casper strain zebrafish N=3 animals n=3 782 cells total. From this we identified ten distinct hematopoietic groups of blood and immune identities. Thirdly we confirmed blood lineage interpretations by comparing hematopoietic lineages within wild type fish with mutant zebrafish with known immunodeficiencies including prkdcD3612fs N=3 animals n=3 201 cells il2rgaY91fs N=2 animals n=2 068 cells and prkdcD3612fs il2rgaY91fs double compound mutant fish N=2 animals n=2 276 cells. Lastly we identified seven structural and functional cell lineages of kidney identities in the whole kidney marrow n=1 699 kidney cells.", "parent bioproject:PRJNA393415", "pubmed:28878000", null, "inDrop sequencing of WT sample animal #2", "GSM2696100", null, "source name:Whole kidney marrow|indrop version:inDrop V2 protocol|tissue:kidney marrow|genotype:wildtype", "inDrop sequencing of WT sample animal #2", "Reads were processed using the indrops pipelinehttps://github.com/indrops/indrops. Briefly reads were filtered according to structure and quality criteria. Filtered reads were then they were sorted by barcode. Demultiplexed reads were aligned to the GRCz10 transcriptome using Bowtie. Please see file \"inDrop GEO supplementary.xlsx \" available on the series record for information on de multiplexing and barcodes for individual samples and cells. Genome build: GRCz10 Supplementary files format and content: text file with read counts.", "Whole kidney marrow", null, "RNA from individual cells were reverse transcribed barcoded and unique transcripts are imprinted with UMI all within the droplet. RNA from 1 500 cells of each animal were pooled and subsequently processed together. cDNA products were pre amplified to produce a cDNA library compatible with the Illumina Nextseq platform. Three sequencing runs were completed using the Nextseq 500 High Output V2 kit 75 cycles Illumina on a NextSeq 500 platform Illumina. Zebrafish cells are isolated from the kidney marrow into single cell suspensions and treated with 15% Optiprep Sigma. Single cells are microfluidically sorted into droplets containing reverse transcription and barcoding reagents. This method is outlined in Klein et al. PMID 26000487 and Zilionis et al. PMID 27929523.", null, "indrop version:inDrop V2 protocol|tissue:kidney marrow|genotype:wildtype", "GSM2696100", "GSM2696100: inDrop sequencing of WT sample animal #2; Danio rerio; RNA Seq", "GSM2696100", null, "1", "RNA from individual cells were reverse transcribed barcoded and unique transcripts are imprinted with UMI all within the droplet. RNA from 1 500 cells of each animal were pooled and subsequently processed together. cDNA products were pre amplified to produce a cDNA library compatible with the Illumina Nextseq platform. Three sequencing runs were completed using the Nextseq 500 High Output V2 kit 75 cycles Illumina on a NextSeq 500 platform Illumina. Zebrafish cells are isolated from the kidney marrow into single cell suspensions and treated with 15% Optiprep Sigma. Single cells are microfluidically sorted into droplets containing reverse transcription and barcoding reagents. This method is outlined in Klein et al. PMID 26000487 and Zilionis et al. PMID 27929523.", "GEO Accession:GSM2696100", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP111339", null, null, "WT2_R2.fastq.gz", "fastq", 4447281250.0, 88945625.0, "GSM2696100 r2", "0:0 1:50", "A:880464480;C:827380868;G:1171423978;T:1561925150;N:6086774", 0, 50, null, null, 880464480, 827380868, 1171423978, 1561925150, 6086774, "SRX2989233", "SRS2341156", "SRA584587", "GEO", "Pathology, Massachusetts General Hospital", 1, 0.00782, null, 0.0075, null, 0.99947, null, 0.91666, null, 50, null, "T", null, "under 1.2% mapping rate", "illumina", "nextseq", "unknown", "random_priming", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-07-07", "Undetermined", "Adult", "Kidney", "Renal System"], [42613, "SRR5810670", "SRX2989232", "SRS2341155", "SRP111339", "PRJNA393429", "Dissecting hematopoietic and renal cell heterogeneity in adult zebrafish at single cell resolution using RNA sequencing [inDrops]", "GSE100910", "Transcriptome Analysis", "Recent advances in single cell transcriptomic profiling have provided unprecedented access to investigate cell heterogeneity during tissue and organ development. Here we utilized massively parallel single cell RNA sequencing to define cell heterogeneity within the zebrafish kidney marrow constructing a comprehensive molecular atlas of definitive hematopoiesis and functionally distinct renal cells found in adult zebrafish. Because our method analyzed blood and kidney cells in an unbiased manner our approach was useful in characterizing immune cell deficiencies within prkdcD3612fs il2rgaY91fs and double homozygous mutant fish identifying blood cell losses in T B and natural killer cells within specific genetic mutants. Our analysis also uncovered novel cell types including two classes of natural killer immune cells classically defined and erythroid primed hematopoietic stem and progenitor cells mucin secreting kidney cells and kidney stem/progenitor cells. In total our work provides the first comprehensive single cell transcriptomic analysis of kidney and marrow cells in the adult zebrafish. Overall design: The goal of our study is to establish the transcriptional profiles of hematopoietic and kidney cell lineages residing in the zebrafish whole kidney marrow. Firstly we performed single cell RNA sequencing by a modified Smart seq2 protocol on sorted single cells from fluorescent transgenic zebrafish lines which label distinct blood cell types n = 246 cells total. Secondly we utilized droplet based single cell RNA sequencing inDrop to investigate unmarked comprehensive hematopoietic lineage structure within wild type casper strain zebrafish N=3 animals n=3 782 cells total. From this we identified ten distinct hematopoietic groups of blood and immune identities. Thirdly we confirmed blood lineage interpretations by comparing hematopoietic lineages within wild type fish with mutant zebrafish with known immunodeficiencies including prkdcD3612fs N=3 animals n=3 201 cells il2rgaY91fs N=2 animals n=2 068 cells and prkdcD3612fs il2rgaY91fs double compound mutant fish N=2 animals n=2 276 cells. Lastly we identified seven structural and functional cell lineages of kidney identities in the whole kidney marrow n=1 699 kidney cells.", "parent bioproject:PRJNA393415", "pubmed:28878000", null, "inDrop sequencing of WT sample animal #1", "GSM2696099", null, "source name:Whole kidney marrow|indrop version:inDrop V2 protocol|tissue:kidney marrow|genotype:wildtype", "inDrop sequencing of WT sample animal #1", "Reads were processed using the indrops pipelinehttps://github.com/indrops/indrops. Briefly reads were filtered according to structure and quality criteria. Filtered reads were then they were sorted by barcode. Demultiplexed reads were aligned to the GRCz10 transcriptome using Bowtie. Please see file \"inDrop GEO supplementary.xlsx \" available on the series record for information on de multiplexing and barcodes for individual samples and cells. Genome build: GRCz10 Supplementary files format and content: text file with read counts.", "Whole kidney marrow", null, "RNA from individual cells were reverse transcribed barcoded and unique transcripts are imprinted with UMI all within the droplet. RNA from 1 500 cells of each animal were pooled and subsequently processed together. cDNA products were pre amplified to produce a cDNA library compatible with the Illumina Nextseq platform. Three sequencing runs were completed using the Nextseq 500 High Output V2 kit 75 cycles Illumina on a NextSeq 500 platform Illumina. Zebrafish cells are isolated from the kidney marrow into single cell suspensions and treated with 15% Optiprep Sigma. Single cells are microfluidically sorted into droplets containing reverse transcription and barcoding reagents. This method is outlined in Klein et al. PMID 26000487 and Zilionis et al. PMID 27929523.", null, "indrop version:inDrop V2 protocol|tissue:kidney marrow|genotype:wildtype", "GSM2696099", "GSM2696099: inDrop sequencing of WT sample animal #1; Danio rerio; RNA Seq", "GSM2696099", null, "1", "RNA from individual cells were reverse transcribed barcoded and unique transcripts are imprinted with UMI all within the droplet. RNA from 1 500 cells of each animal were pooled and subsequently processed together. cDNA products were pre amplified to produce a cDNA library compatible with the Illumina Nextseq platform. Three sequencing runs were completed using the Nextseq 500 High Output V2 kit 75 cycles Illumina on a NextSeq 500 platform Illumina. Zebrafish cells are isolated from the kidney marrow into single cell suspensions and treated with 15% Optiprep Sigma. Single cells are microfluidically sorted into droplets containing reverse transcription and barcoding reagents. This method is outlined in Klein et al. PMID 26000487 and Zilionis et al. PMID 27929523.", "GEO Accession:GSM2696099", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP111339", null, null, "WT1_R1.fastq.gz", "fastq", 3313205496.0, 92033486.0, "GSM2696099 r1", "0:36 1:0", "A:858288917;C:693848400;G:675650809;T:1085369048;N:48322", 36, 0, null, null, 858288917, 693848400, 675650809, 1085369048, 48322, "SRX2989232", "SRS2341155", "SRA584587", "GEO", "Pathology, Massachusetts General Hospital", 1, 0.8771, null, 0.13918, null, 0.81115, null, 0.59004, null, 36, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "random_priming", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-07-07", "Undetermined", "Adult", "Kidney", "Renal System"], [42614, "SRR5810671", "SRX2989232", "SRS2341155", "SRP111339", "PRJNA393429", "Dissecting hematopoietic and renal cell heterogeneity in adult zebrafish at single cell resolution using RNA sequencing [inDrops]", "GSE100910", "Transcriptome Analysis", "Recent advances in single cell transcriptomic profiling have provided unprecedented access to investigate cell heterogeneity during tissue and organ development. Here we utilized massively parallel single cell RNA sequencing to define cell heterogeneity within the zebrafish kidney marrow constructing a comprehensive molecular atlas of definitive hematopoiesis and functionally distinct renal cells found in adult zebrafish. Because our method analyzed blood and kidney cells in an unbiased manner our approach was useful in characterizing immune cell deficiencies within prkdcD3612fs il2rgaY91fs and double homozygous mutant fish identifying blood cell losses in T B and natural killer cells within specific genetic mutants. Our analysis also uncovered novel cell types including two classes of natural killer immune cells classically defined and erythroid primed hematopoietic stem and progenitor cells mucin secreting kidney cells and kidney stem/progenitor cells. In total our work provides the first comprehensive single cell transcriptomic analysis of kidney and marrow cells in the adult zebrafish. Overall design: The goal of our study is to establish the transcriptional profiles of hematopoietic and kidney cell lineages residing in the zebrafish whole kidney marrow. Firstly we performed single cell RNA sequencing by a modified Smart seq2 protocol on sorted single cells from fluorescent transgenic zebrafish lines which label distinct blood cell types n = 246 cells total. Secondly we utilized droplet based single cell RNA sequencing inDrop to investigate unmarked comprehensive hematopoietic lineage structure within wild type casper strain zebrafish N=3 animals n=3 782 cells total. From this we identified ten distinct hematopoietic groups of blood and immune identities. Thirdly we confirmed blood lineage interpretations by comparing hematopoietic lineages within wild type fish with mutant zebrafish with known immunodeficiencies including prkdcD3612fs N=3 animals n=3 201 cells il2rgaY91fs N=2 animals n=2 068 cells and prkdcD3612fs il2rgaY91fs double compound mutant fish N=2 animals n=2 276 cells. Lastly we identified seven structural and functional cell lineages of kidney identities in the whole kidney marrow n=1 699 kidney cells.", "parent bioproject:PRJNA393415", "pubmed:28878000", null, "inDrop sequencing of WT sample animal #1", "GSM2696099", null, "source name:Whole kidney marrow|indrop version:inDrop V2 protocol|tissue:kidney marrow|genotype:wildtype", "inDrop sequencing of WT sample animal #1", "Reads were processed using the indrops pipelinehttps://github.com/indrops/indrops. Briefly reads were filtered according to structure and quality criteria. Filtered reads were then they were sorted by barcode. Demultiplexed reads were aligned to the GRCz10 transcriptome using Bowtie. Please see file \"inDrop GEO supplementary.xlsx \" available on the series record for information on de multiplexing and barcodes for individual samples and cells. Genome build: GRCz10 Supplementary files format and content: text file with read counts.", "Whole kidney marrow", null, "RNA from individual cells were reverse transcribed barcoded and unique transcripts are imprinted with UMI all within the droplet. RNA from 1 500 cells of each animal were pooled and subsequently processed together. cDNA products were pre amplified to produce a cDNA library compatible with the Illumina Nextseq platform. Three sequencing runs were completed using the Nextseq 500 High Output V2 kit 75 cycles Illumina on a NextSeq 500 platform Illumina. Zebrafish cells are isolated from the kidney marrow into single cell suspensions and treated with 15% Optiprep Sigma. Single cells are microfluidically sorted into droplets containing reverse transcription and barcoding reagents. This method is outlined in Klein et al. PMID 26000487 and Zilionis et al. PMID 27929523.", null, "indrop version:inDrop V2 protocol|tissue:kidney marrow|genotype:wildtype", "GSM2696099", "GSM2696099: inDrop sequencing of WT sample animal #1; Danio rerio; RNA Seq", "GSM2696099", null, "1", "RNA from individual cells were reverse transcribed barcoded and unique transcripts are imprinted with UMI all within the droplet. RNA from 1 500 cells of each animal were pooled and subsequently processed together. cDNA products were pre amplified to produce a cDNA library compatible with the Illumina Nextseq platform. Three sequencing runs were completed using the Nextseq 500 High Output V2 kit 75 cycles Illumina on a NextSeq 500 platform Illumina. Zebrafish cells are isolated from the kidney marrow into single cell suspensions and treated with 15% Optiprep Sigma. Single cells are microfluidically sorted into droplets containing reverse transcription and barcoding reagents. This method is outlined in Klein et al. PMID 26000487 and Zilionis et al. PMID 27929523.", "GEO Accession:GSM2696099", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP111339", null, null, "WT1_R2.fastq.gz", "fastq", 4601674300.0, 92033486.0, "GSM2696099 r2", "0:0 1:50", "A:914884703;C:856837987;G:1192418010;T:1631240786;N:6292814", 0, 50, null, null, 914884703, 856837987, 1192418010, 1631240786, 6292814, "SRX2989232", "SRS2341155", "SRA584587", "GEO", "Pathology, Massachusetts General Hospital", 1, 0.0063, null, 0.00598, null, 0.99959, null, 0.8913, null, 50, null, "T", null, "under 1.2% mapping rate", "illumina", "nextseq", "unknown", "random_priming", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-07-07", "Undetermined", "Adult", "Kidney", "Renal System"], [43842, "SRR6176750", "SRX3287416", "SRS2596889", "SRP120009", "PRJNA414416", "Simultaneous single cell profiling of lineages and cell types in the vertebrate brain", "GSE105010", "Other", "The lineage relationships among the hundreds of cell types generated during development are difficult to reconstruct. A recent method GESTALT used CRISPR\u2013Cas9 barcode editing for large scale lineage tracing but was restricted to early development and did not identify cell types. Here we present scGESTALT which combines the lineage recording capabilities of GESTALT with cell type identification by single cell RNA sequencing. The method relies on an inducible system that enables barcodes to be edited at multiple time points capturing lineage information from later stages of development. Sequencing of 60 000 transcriptomes from the juvenile zebrafish brain identified >100 cell types and marker genes. Using these data we generate lineage trees with hundreds of branches that help uncover restrictions at the level of cell types brain regions and gene expression cascades during differentiation. scGESTALT can be applied to other multicellular organisms to simultaneously characterize molecular identities and lineage histories of thousands of cells during development and disease. Overall design: inDrops libraries of single cell transcriptomes and scGESTALT barcodes and genomic DNA GESTALT libraries", null, "pubmed:29608178", null, "ZF3 scGSTLT", "GSM2813986", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "ZF3 scGSTLT", "Single cell RNA Sequencing data FASTQ files were processed using the inDrops.py bioinformatics pipeline available at https://github.com/indrops/indrops. Transcriptome libraries were mapped to a zebrafish reference built from a custom GTF file and the zebrafish GRCz10 release 86 genome assembly. Bowtie version1.1.1 was used with parameter \u2013e 200; UMI quantification was used with parameter \u2013u 2 counts were ignored from UMIs split between more than 2 genes. genomic DNA GESTALT and scGESTALT libraries were processed using a custom pipeline available at https://github.com/shendurelab/Cas9FateMapping Genome build: GRCz10 CSV files for transcriptome data were generated using the inDrops pipeline. Each column in the CSV files contains a cell identifier and each row contains expression values for genes. Txt files for genomic DNA GESTALT libraries *allReadCounts contain lineage barcode sequences HMID column for each cell and their proportion in the sequenced libraries. scGESTALT data *GestMaster.txt contains the inDrops cell identifiers CellBarcode and BarcodeKey that were used to match barcodes to transcriptomes. They also contain lineage barcode sequences HMID column for each cell with a corresponding inDrops single cell gene expression profile as well as the the t SNE cluster membership number ClusterIdent column Txt files ending in *stats.txt contain information about the each individually captured UMI or cell per sample. The barcode sequence aligned to a reference unedited sequence mergedRead column mutations at each target site target[X] columns and the edited sequences at each target site sequence[X] columns were used for downstream analysis. inDropsExpMatrix noQ txt file is the gene expression matrix for the full dataset. Columns are individual cells from different batches of whole brains f1 f2 f3 f4 f5 f6 or brain regions fore mid hind . fall.inDrops.Robj is the processed Seurat R object which can be loaded into R and explored.", "zebrafish brain", null, "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed reverse transcribed and prepared for sequencing Gestalt barcode was PCR amplified by two step PCR. Sample indices and flow cell adaptors were then added by PCR.", null, "tissue:brain|developmental stage:23 25dpf", "GSM2813986", "GSM2813986: ZF3 scGSTLT; Danio rerio; OTHER", "GSM2813986", null, "1", "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed reverse transcribed and prepared for sequencing Gestalt barcode was PCR amplified by two step PCR. Sample indices and flow cell adaptors were then added by PCR.", "GEO Accession:GSM2813986", "OTHER", "TRANSCRIPTOMIC", "other", "SINGLE", "ILLUMINA", "Illumina MiSeq", null, "SRP120009", null, "loader:fastq load.py|options: appendBCtoName", "F6_UMI.merged.fq.gz", "fastq", 786192951.0, 2901081.0, "GSM2813986 r1", "0:271", "A:215364033;C:176865797;G:224444690;T:169518396;N:35", 271, null, null, null, 215364033, 176865797, 224444690, 169518396, 35, "SRX3287416", "SRS2596889", "SRA619743", "GEO", "Harvard University", 1, 0.00904, null, 0.0, null, 0.99997, null, 0.0, null, 271, null, "T", null, "under 1.2% mapping rate", "illumina", "miseq", "unknown", "other", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43843, "SRR6176749", "SRX3287415", "SRS2596888", "SRP120009", "PRJNA414416", "Simultaneous single cell profiling of lineages and cell types in the vertebrate brain", "GSE105010", "Other", "The lineage relationships among the hundreds of cell types generated during development are difficult to reconstruct. A recent method GESTALT used CRISPR\u2013Cas9 barcode editing for large scale lineage tracing but was restricted to early development and did not identify cell types. Here we present scGESTALT which combines the lineage recording capabilities of GESTALT with cell type identification by single cell RNA sequencing. The method relies on an inducible system that enables barcodes to be edited at multiple time points capturing lineage information from later stages of development. Sequencing of 60 000 transcriptomes from the juvenile zebrafish brain identified >100 cell types and marker genes. Using these data we generate lineage trees with hundreds of branches that help uncover restrictions at the level of cell types brain regions and gene expression cascades during differentiation. scGESTALT can be applied to other multicellular organisms to simultaneously characterize molecular identities and lineage histories of thousands of cells during development and disease. Overall design: inDrops libraries of single cell transcriptomes and scGESTALT barcodes and genomic DNA GESTALT libraries", null, "pubmed:29608178", null, "ZF2 scGSTLT", "GSM2813985", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "ZF2 scGSTLT", "Single cell RNA Sequencing data FASTQ files were processed using the inDrops.py bioinformatics pipeline available at https://github.com/indrops/indrops. Transcriptome libraries were mapped to a zebrafish reference built from a custom GTF file and the zebrafish GRCz10 release 86 genome assembly. Bowtie version1.1.1 was used with parameter \u2013e 200; UMI quantification was used with parameter \u2013u 2 counts were ignored from UMIs split between more than 2 genes. genomic DNA GESTALT and scGESTALT libraries were processed using a custom pipeline available at https://github.com/shendurelab/Cas9FateMapping Genome build: GRCz10 CSV files for transcriptome data were generated using the inDrops pipeline. Each column in the CSV files contains a cell identifier and each row contains expression values for genes. Txt files for genomic DNA GESTALT libraries *allReadCounts contain lineage barcode sequences HMID column for each cell and their proportion in the sequenced libraries. scGESTALT data *GestMaster.txt contains the inDrops cell identifiers CellBarcode and BarcodeKey that were used to match barcodes to transcriptomes. They also contain lineage barcode sequences HMID column for each cell with a corresponding inDrops single cell gene expression profile as well as the the t SNE cluster membership number ClusterIdent column Txt files ending in *stats.txt contain information about the each individually captured UMI or cell per sample. The barcode sequence aligned to a reference unedited sequence mergedRead column mutations at each target site target[X] columns and the edited sequences at each target site sequence[X] columns were used for downstream analysis. inDropsExpMatrix noQ txt file is the gene expression matrix for the full dataset. Columns are individual cells from different batches of whole brains f1 f2 f3 f4 f5 f6 or brain regions fore mid hind . fall.inDrops.Robj is the processed Seurat R object which can be loaded into R and explored.", "zebrafish brain", null, "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed reverse transcribed and prepared for sequencing Gestalt barcode was PCR amplified by two step PCR. Sample indices and flow cell adaptors were then added by PCR.", null, "tissue:brain|developmental stage:23 25dpf", "GSM2813985", "GSM2813985: ZF2 scGSTLT; Danio rerio; OTHER", "GSM2813985", null, "1", "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed reverse transcribed and prepared for sequencing Gestalt barcode was PCR amplified by two step PCR. Sample indices and flow cell adaptors were then added by PCR.", "GEO Accession:GSM2813985", "OTHER", "TRANSCRIPTOMIC", "other", "SINGLE", "ILLUMINA", "Illumina MiSeq", null, "SRP120009", null, "loader:fastq load.py|options: appendBCtoName", "F5_UMI.merged.fq.gz", "fastq", 106145626.0, 391949.0, "GSM2813985 r1", "0:270.81", "A:29282656;C:22944139;G:29276360;T:24642464;N:7", 270, null, null, null, 29282656, 22944139, 29276360, 24642464, 7, "SRX3287415", "SRS2596888", "SRA619743", "GEO", "Harvard University", 1, 2e-05, null, 0.0, null, 0.99995, null, 0.0, null, 270, null, "T", null, "under 1.2% mapping rate", "illumina", "miseq", "unknown", "other", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43844, "SRR6176748", "SRX3287414", "SRS2596887", "SRP120009", "PRJNA414416", "Simultaneous single cell profiling of lineages and cell types in the vertebrate brain", "GSE105010", "Other", "The lineage relationships among the hundreds of cell types generated during development are difficult to reconstruct. A recent method GESTALT used CRISPR\u2013Cas9 barcode editing for large scale lineage tracing but was restricted to early development and did not identify cell types. Here we present scGESTALT which combines the lineage recording capabilities of GESTALT with cell type identification by single cell RNA sequencing. The method relies on an inducible system that enables barcodes to be edited at multiple time points capturing lineage information from later stages of development. Sequencing of 60 000 transcriptomes from the juvenile zebrafish brain identified >100 cell types and marker genes. Using these data we generate lineage trees with hundreds of branches that help uncover restrictions at the level of cell types brain regions and gene expression cascades during differentiation. scGESTALT can be applied to other multicellular organisms to simultaneously characterize molecular identities and lineage histories of thousands of cells during development and disease. Overall design: inDrops libraries of single cell transcriptomes and scGESTALT barcodes and genomic DNA GESTALT libraries", null, "pubmed:29608178", null, "ZF1 scGSTLT", "GSM2813984", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "ZF1 scGSTLT", "Single cell RNA Sequencing data FASTQ files were processed using the inDrops.py bioinformatics pipeline available at https://github.com/indrops/indrops. Transcriptome libraries were mapped to a zebrafish reference built from a custom GTF file and the zebrafish GRCz10 release 86 genome assembly. Bowtie version1.1.1 was used with parameter \u2013e 200; UMI quantification was used with parameter \u2013u 2 counts were ignored from UMIs split between more than 2 genes. genomic DNA GESTALT and scGESTALT libraries were processed using a custom pipeline available at https://github.com/shendurelab/Cas9FateMapping Genome build: GRCz10 CSV files for transcriptome data were generated using the inDrops pipeline. Each column in the CSV files contains a cell identifier and each row contains expression values for genes. Txt files for genomic DNA GESTALT libraries *allReadCounts contain lineage barcode sequences HMID column for each cell and their proportion in the sequenced libraries. scGESTALT data *GestMaster.txt contains the inDrops cell identifiers CellBarcode and BarcodeKey that were used to match barcodes to transcriptomes. They also contain lineage barcode sequences HMID column for each cell with a corresponding inDrops single cell gene expression profile as well as the the t SNE cluster membership number ClusterIdent column Txt files ending in *stats.txt contain information about the each individually captured UMI or cell per sample. The barcode sequence aligned to a reference unedited sequence mergedRead column mutations at each target site target[X] columns and the edited sequences at each target site sequence[X] columns were used for downstream analysis. inDropsExpMatrix noQ txt file is the gene expression matrix for the full dataset. Columns are individual cells from different batches of whole brains f1 f2 f3 f4 f5 f6 or brain regions fore mid hind . fall.inDrops.Robj is the processed Seurat R object which can be loaded into R and explored.", "zebrafish brain", null, "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed reverse transcribed and prepared for sequencing Gestalt barcode was PCR amplified by two step PCR. Sample indices and flow cell adaptors were then added by PCR.", null, "tissue:brain|developmental stage:23 25dpf", "GSM2813984", "GSM2813984: ZF1 scGSTLT; Danio rerio; OTHER", "GSM2813984", null, "1", "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed reverse transcribed and prepared for sequencing Gestalt barcode was PCR amplified by two step PCR. Sample indices and flow cell adaptors were then added by PCR.", "GEO Accession:GSM2813984", "OTHER", "TRANSCRIPTOMIC", "other", "SINGLE", "ILLUMINA", "Illumina MiSeq", null, "SRP120009", null, "loader:fastq load.py|options: appendBCtoName", "F3_UMI.merged.fq.gz", "fastq", 817055786.0, 3014966.0, "GSM2813984 r1", "0:271", "A:235846657;C:170311176;G:222336783;T:188561170;N:0", 271, null, null, null, 235846657, 170311176, 222336783, 188561170, 0, "SRX3287414", "SRS2596887", "SRA619743", "GEO", "Harvard University", 1, 4e-05, null, 0.0, null, 0.99995, null, 0.4, null, 271, null, "T", null, "under 1.2% mapping rate", "illumina", "miseq", "unknown", "other", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43845, "SRR6176746", "SRX3287413", "SRS2596886", "SRP120009", "PRJNA414416", "Simultaneous single cell profiling of lineages and cell types in the vertebrate brain", "GSE105010", "Other", "The lineage relationships among the hundreds of cell types generated during development are difficult to reconstruct. A recent method GESTALT used CRISPR\u2013Cas9 barcode editing for large scale lineage tracing but was restricted to early development and did not identify cell types. Here we present scGESTALT which combines the lineage recording capabilities of GESTALT with cell type identification by single cell RNA sequencing. The method relies on an inducible system that enables barcodes to be edited at multiple time points capturing lineage information from later stages of development. Sequencing of 60 000 transcriptomes from the juvenile zebrafish brain identified >100 cell types and marker genes. Using these data we generate lineage trees with hundreds of branches that help uncover restrictions at the level of cell types brain regions and gene expression cascades during differentiation. scGESTALT can be applied to other multicellular organisms to simultaneously characterize molecular identities and lineage histories of thousands of cells during development and disease. Overall design: inDrops libraries of single cell transcriptomes and scGESTALT barcodes and genomic DNA GESTALT libraries", null, "pubmed:29608178", null, "DEW157 mid", "GSM2813983", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW157 mid", "Single cell RNA Sequencing data FASTQ files were processed using the inDrops.py bioinformatics pipeline available at https://github.com/indrops/indrops. Transcriptome libraries were mapped to a zebrafish reference built from a custom GTF file and the zebrafish GRCz10 release 86 genome assembly. Bowtie version1.1.1 was used with parameter \u2013e 200; UMI quantification was used with parameter \u2013u 2 counts were ignored from UMIs split between more than 2 genes. genomic DNA GESTALT and scGESTALT libraries were processed using a custom pipeline available at https://github.com/shendurelab/Cas9FateMapping Genome build: GRCz10 CSV files for transcriptome data were generated using the inDrops pipeline. Each column in the CSV files contains a cell identifier and each row contains expression values for genes. Txt files for genomic DNA GESTALT libraries *allReadCounts contain lineage barcode sequences HMID column for each cell and their proportion in the sequenced libraries. scGESTALT data *GestMaster.txt contains the inDrops cell identifiers CellBarcode and BarcodeKey that were used to match barcodes to transcriptomes. They also contain lineage barcode sequences HMID column for each cell with a corresponding inDrops single cell gene expression profile as well as the the t SNE cluster membership number ClusterIdent column Txt files ending in *stats.txt contain information about the each individually captured UMI or cell per sample. The barcode sequence aligned to a reference unedited sequence mergedRead column mutations at each target site target[X] columns and the edited sequences at each target site sequence[X] columns were used for downstream analysis. inDropsExpMatrix noQ txt file is the gene expression matrix for the full dataset. Columns are individual cells from different batches of whole brains f1 f2 f3 f4 f5 f6 or brain regions fore mid hind . fall.inDrops.Robj is the processed Seurat R object which can be loaded into R and explored.", "zebrafish brain", null, "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed and fragmented. The three prime fragments were reverse transcribed and prepared for sequencing Libraries were prepared as described in Zilionis et al. 2017 Nature Protocols PMID = 27929523", null, "tissue:brain|developmental stage:23 25dpf", "GSM2813983", "GSM2813983: DEW157 mid; Danio rerio; RNA Seq", "GSM2813983", null, "1", "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed and fragmented. The three prime fragments were reverse transcribed and prepared for sequencing Libraries were prepared as described in Zilionis et al. 2017 Nature Protocols PMID = 27929523", "GEO Accession:GSM2813983", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options: appendBCtoName", "DEW157_Lane1.sorted.fastq.gz", "fastq", 3544126799.0, 61301471.0, "GSM2813983 r1", "0:57.81", "A:974428815;C:692114455;G:739762074;T:1137804675;N:16780", 57, null, null, null, 974428815, 692114455, 739762074, 1137804675, 16780, "SRX3287413", "SRS2596886", "SRA619743", "GEO", "Harvard University", 1, 0.89639, null, 0.20572, null, 0.77784, null, 0.5325, null, 61, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43846, "SRR6176747", "SRX3287413", "SRS2596886", "SRP120009", "PRJNA414416", "Simultaneous single cell profiling of lineages and cell types in the vertebrate brain", "GSE105010", "Other", "The lineage relationships among the hundreds of cell types generated during development are difficult to reconstruct. A recent method GESTALT used CRISPR\u2013Cas9 barcode editing for large scale lineage tracing but was restricted to early development and did not identify cell types. Here we present scGESTALT which combines the lineage recording capabilities of GESTALT with cell type identification by single cell RNA sequencing. The method relies on an inducible system that enables barcodes to be edited at multiple time points capturing lineage information from later stages of development. Sequencing of 60 000 transcriptomes from the juvenile zebrafish brain identified >100 cell types and marker genes. Using these data we generate lineage trees with hundreds of branches that help uncover restrictions at the level of cell types brain regions and gene expression cascades during differentiation. scGESTALT can be applied to other multicellular organisms to simultaneously characterize molecular identities and lineage histories of thousands of cells during development and disease. Overall design: inDrops libraries of single cell transcriptomes and scGESTALT barcodes and genomic DNA GESTALT libraries", null, "pubmed:29608178", null, "DEW157 mid", "GSM2813983", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW157 mid", "Single cell RNA Sequencing data FASTQ files were processed using the inDrops.py bioinformatics pipeline available at https://github.com/indrops/indrops. Transcriptome libraries were mapped to a zebrafish reference built from a custom GTF file and the zebrafish GRCz10 release 86 genome assembly. Bowtie version1.1.1 was used with parameter \u2013e 200; UMI quantification was used with parameter \u2013u 2 counts were ignored from UMIs split between more than 2 genes. genomic DNA GESTALT and scGESTALT libraries were processed using a custom pipeline available at https://github.com/shendurelab/Cas9FateMapping Genome build: GRCz10 CSV files for transcriptome data were generated using the inDrops pipeline. Each column in the CSV files contains a cell identifier and each row contains expression values for genes. Txt files for genomic DNA GESTALT libraries *allReadCounts contain lineage barcode sequences HMID column for each cell and their proportion in the sequenced libraries. scGESTALT data *GestMaster.txt contains the inDrops cell identifiers CellBarcode and BarcodeKey that were used to match barcodes to transcriptomes. They also contain lineage barcode sequences HMID column for each cell with a corresponding inDrops single cell gene expression profile as well as the the t SNE cluster membership number ClusterIdent column Txt files ending in *stats.txt contain information about the each individually captured UMI or cell per sample. The barcode sequence aligned to a reference unedited sequence mergedRead column mutations at each target site target[X] columns and the edited sequences at each target site sequence[X] columns were used for downstream analysis. inDropsExpMatrix noQ txt file is the gene expression matrix for the full dataset. Columns are individual cells from different batches of whole brains f1 f2 f3 f4 f5 f6 or brain regions fore mid hind . fall.inDrops.Robj is the processed Seurat R object which can be loaded into R and explored.", "zebrafish brain", null, "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed and fragmented. The three prime fragments were reverse transcribed and prepared for sequencing Libraries were prepared as described in Zilionis et al. 2017 Nature Protocols PMID = 27929523", null, "tissue:brain|developmental stage:23 25dpf", "GSM2813983", "GSM2813983: DEW157 mid; Danio rerio; RNA Seq", "GSM2813983", null, "1", "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed and fragmented. The three prime fragments were reverse transcribed and prepared for sequencing Libraries were prepared as described in Zilionis et al. 2017 Nature Protocols PMID = 27929523", "GEO Accession:GSM2813983", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options: appendBCtoName", "DEW157_Lane2.sorted.fastq.gz", "fastq", 2352087459.0, 41062625.0, "GSM2813983 r2", "0:57.28", "A:645085555;C:458868888;G:493735966;T:754390615;N:6435", 57, null, null, null, 645085555, 458868888, 493735966, 754390615, 6435, "SRX3287413", "SRS2596886", "SRA619743", "GEO", "Harvard University", 1, 0.89716, null, 0.2057, null, 0.7782, null, 0.5213, null, 61, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43847, "SRR6176744", "SRX3287412", "SRS2596885", "SRP120009", "PRJNA414416", "Simultaneous single cell profiling of lineages and cell types in the vertebrate brain", "GSE105010", "Other", "The lineage relationships among the hundreds of cell types generated during development are difficult to reconstruct. A recent method GESTALT used CRISPR\u2013Cas9 barcode editing for large scale lineage tracing but was restricted to early development and did not identify cell types. Here we present scGESTALT which combines the lineage recording capabilities of GESTALT with cell type identification by single cell RNA sequencing. The method relies on an inducible system that enables barcodes to be edited at multiple time points capturing lineage information from later stages of development. Sequencing of 60 000 transcriptomes from the juvenile zebrafish brain identified >100 cell types and marker genes. Using these data we generate lineage trees with hundreds of branches that help uncover restrictions at the level of cell types brain regions and gene expression cascades during differentiation. scGESTALT can be applied to other multicellular organisms to simultaneously characterize molecular identities and lineage histories of thousands of cells during development and disease. Overall design: inDrops libraries of single cell transcriptomes and scGESTALT barcodes and genomic DNA GESTALT libraries", null, "pubmed:29608178", null, "DEW156 mid", "GSM2813982", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW156 mid", "Single cell RNA Sequencing data FASTQ files were processed using the inDrops.py bioinformatics pipeline available at https://github.com/indrops/indrops. Transcriptome libraries were mapped to a zebrafish reference built from a custom GTF file and the zebrafish GRCz10 release 86 genome assembly. Bowtie version1.1.1 was used with parameter \u2013e 200; UMI quantification was used with parameter \u2013u 2 counts were ignored from UMIs split between more than 2 genes. genomic DNA GESTALT and scGESTALT libraries were processed using a custom pipeline available at https://github.com/shendurelab/Cas9FateMapping Genome build: GRCz10 CSV files for transcriptome data were generated using the inDrops pipeline. Each column in the CSV files contains a cell identifier and each row contains expression values for genes. Txt files for genomic DNA GESTALT libraries *allReadCounts contain lineage barcode sequences HMID column for each cell and their proportion in the sequenced libraries. scGESTALT data *GestMaster.txt contains the inDrops cell identifiers CellBarcode and BarcodeKey that were used to match barcodes to transcriptomes. They also contain lineage barcode sequences HMID column for each cell with a corresponding inDrops single cell gene expression profile as well as the the t SNE cluster membership number ClusterIdent column Txt files ending in *stats.txt contain information about the each individually captured UMI or cell per sample. The barcode sequence aligned to a reference unedited sequence mergedRead column mutations at each target site target[X] columns and the edited sequences at each target site sequence[X] columns were used for downstream analysis. inDropsExpMatrix noQ txt file is the gene expression matrix for the full dataset. Columns are individual cells from different batches of whole brains f1 f2 f3 f4 f5 f6 or brain regions fore mid hind . fall.inDrops.Robj is the processed Seurat R object which can be loaded into R and explored.", "zebrafish brain", null, "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed and fragmented. The three prime fragments were reverse transcribed and prepared for sequencing Libraries were prepared as described in Zilionis et al. 2017 Nature Protocols PMID = 27929523", null, "tissue:brain|developmental stage:23 25dpf", "GSM2813982", "GSM2813982: DEW156 mid; Danio rerio; RNA Seq", "GSM2813982", null, "1", "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed and fragmented. The three prime fragments were reverse transcribed and prepared for sequencing Libraries were prepared as described in Zilionis et al. 2017 Nature Protocols PMID = 27929523", "GEO Accession:GSM2813982", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options: appendBCtoName", "DEW156_Lane1.sorted.fastq.gz", "fastq", 2214394241.0, 38215249.0, "GSM2813982 r1", "0:57.95", "A:607624943;C:431025702;G:463801442;T:711931460;N:10694", 57, null, null, null, 607624943, 431025702, 463801442, 711931460, 10694, "SRX3287412", "SRS2596885", "SRA619743", "GEO", "Harvard University", 1, 0.89922, null, 0.20797, null, 0.77626, null, 0.52363, null, 61, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43848, "SRR6176745", "SRX3287412", "SRS2596885", "SRP120009", "PRJNA414416", "Simultaneous single cell profiling of lineages and cell types in the vertebrate brain", "GSE105010", "Other", "The lineage relationships among the hundreds of cell types generated during development are difficult to reconstruct. A recent method GESTALT used CRISPR\u2013Cas9 barcode editing for large scale lineage tracing but was restricted to early development and did not identify cell types. Here we present scGESTALT which combines the lineage recording capabilities of GESTALT with cell type identification by single cell RNA sequencing. The method relies on an inducible system that enables barcodes to be edited at multiple time points capturing lineage information from later stages of development. Sequencing of 60 000 transcriptomes from the juvenile zebrafish brain identified >100 cell types and marker genes. Using these data we generate lineage trees with hundreds of branches that help uncover restrictions at the level of cell types brain regions and gene expression cascades during differentiation. scGESTALT can be applied to other multicellular organisms to simultaneously characterize molecular identities and lineage histories of thousands of cells during development and disease. Overall design: inDrops libraries of single cell transcriptomes and scGESTALT barcodes and genomic DNA GESTALT libraries", null, "pubmed:29608178", null, "DEW156 mid", "GSM2813982", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW156 mid", "Single cell RNA Sequencing data FASTQ files were processed using the inDrops.py bioinformatics pipeline available at https://github.com/indrops/indrops. Transcriptome libraries were mapped to a zebrafish reference built from a custom GTF file and the zebrafish GRCz10 release 86 genome assembly. Bowtie version1.1.1 was used with parameter \u2013e 200; UMI quantification was used with parameter \u2013u 2 counts were ignored from UMIs split between more than 2 genes. genomic DNA GESTALT and scGESTALT libraries were processed using a custom pipeline available at https://github.com/shendurelab/Cas9FateMapping Genome build: GRCz10 CSV files for transcriptome data were generated using the inDrops pipeline. Each column in the CSV files contains a cell identifier and each row contains expression values for genes. Txt files for genomic DNA GESTALT libraries *allReadCounts contain lineage barcode sequences HMID column for each cell and their proportion in the sequenced libraries. scGESTALT data *GestMaster.txt contains the inDrops cell identifiers CellBarcode and BarcodeKey that were used to match barcodes to transcriptomes. They also contain lineage barcode sequences HMID column for each cell with a corresponding inDrops single cell gene expression profile as well as the the t SNE cluster membership number ClusterIdent column Txt files ending in *stats.txt contain information about the each individually captured UMI or cell per sample. The barcode sequence aligned to a reference unedited sequence mergedRead column mutations at each target site target[X] columns and the edited sequences at each target site sequence[X] columns were used for downstream analysis. inDropsExpMatrix noQ txt file is the gene expression matrix for the full dataset. Columns are individual cells from different batches of whole brains f1 f2 f3 f4 f5 f6 or brain regions fore mid hind . fall.inDrops.Robj is the processed Seurat R object which can be loaded into R and explored.", "zebrafish brain", null, "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed and fragmented. The three prime fragments were reverse transcribed and prepared for sequencing Libraries were prepared as described in Zilionis et al. 2017 Nature Protocols PMID = 27929523", null, "tissue:brain|developmental stage:23 25dpf", "GSM2813982", "GSM2813982: DEW156 mid; Danio rerio; RNA Seq", "GSM2813982", null, "1", "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed and fragmented. The three prime fragments were reverse transcribed and prepared for sequencing Libraries were prepared as described in Zilionis et al. 2017 Nature Protocols PMID = 27929523", "GEO Accession:GSM2813982", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options: appendBCtoName", "DEW156_Lane2.sorted.fastq.gz", "fastq", 1448314433.0, 25210929.0, "GSM2813982 r2", "0:57.45", "A:396430204;C:281605460;G:305048301;T:465226358;N:4110", 57, null, null, null, 396430204, 281605460, 305048301, 465226358, 4110, "SRX3287412", "SRS2596885", "SRA619743", "GEO", "Harvard University", 1, 0.90054, null, 0.21045, null, 0.77508, null, 0.51559, null, 61, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43849, "SRR6176742", "SRX3287411", "SRS2596902", "SRP120009", "PRJNA414416", "Simultaneous single cell profiling of lineages and cell types in the vertebrate brain", "GSE105010", "Other", "The lineage relationships among the hundreds of cell types generated during development are difficult to reconstruct. A recent method GESTALT used CRISPR\u2013Cas9 barcode editing for large scale lineage tracing but was restricted to early development and did not identify cell types. Here we present scGESTALT which combines the lineage recording capabilities of GESTALT with cell type identification by single cell RNA sequencing. The method relies on an inducible system that enables barcodes to be edited at multiple time points capturing lineage information from later stages of development. Sequencing of 60 000 transcriptomes from the juvenile zebrafish brain identified >100 cell types and marker genes. Using these data we generate lineage trees with hundreds of branches that help uncover restrictions at the level of cell types brain regions and gene expression cascades during differentiation. scGESTALT can be applied to other multicellular organisms to simultaneously characterize molecular identities and lineage histories of thousands of cells during development and disease. Overall design: inDrops libraries of single cell transcriptomes and scGESTALT barcodes and genomic DNA GESTALT libraries", null, "pubmed:29608178", null, "DEW155 mid", "GSM2813981", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW155 mid", "Single cell RNA Sequencing data FASTQ files were processed using the inDrops.py bioinformatics pipeline available at https://github.com/indrops/indrops. Transcriptome libraries were mapped to a zebrafish reference built from a custom GTF file and the zebrafish GRCz10 release 86 genome assembly. Bowtie version1.1.1 was used with parameter \u2013e 200; UMI quantification was used with parameter \u2013u 2 counts were ignored from UMIs split between more than 2 genes. genomic DNA GESTALT and scGESTALT libraries were processed using a custom pipeline available at https://github.com/shendurelab/Cas9FateMapping Genome build: GRCz10 CSV files for transcriptome data were generated using the inDrops pipeline. Each column in the CSV files contains a cell identifier and each row contains expression values for genes. Txt files for genomic DNA GESTALT libraries *allReadCounts contain lineage barcode sequences HMID column for each cell and their proportion in the sequenced libraries. scGESTALT data *GestMaster.txt contains the inDrops cell identifiers CellBarcode and BarcodeKey that were used to match barcodes to transcriptomes. They also contain lineage barcode sequences HMID column for each cell with a corresponding inDrops single cell gene expression profile as well as the the t SNE cluster membership number ClusterIdent column Txt files ending in *stats.txt contain information about the each individually captured UMI or cell per sample. The barcode sequence aligned to a reference unedited sequence mergedRead column mutations at each target site target[X] columns and the edited sequences at each target site sequence[X] columns were used for downstream analysis. inDropsExpMatrix noQ txt file is the gene expression matrix for the full dataset. Columns are individual cells from different batches of whole brains f1 f2 f3 f4 f5 f6 or brain regions fore mid hind . fall.inDrops.Robj is the processed Seurat R object which can be loaded into R and explored.", "zebrafish brain", null, "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed and fragmented. The three prime fragments were reverse transcribed and prepared for sequencing Libraries were prepared as described in Zilionis et al. 2017 Nature Protocols PMID = 27929523", null, "tissue:brain|developmental stage:23 25dpf", "GSM2813981", "GSM2813981: DEW155 mid; Danio rerio; RNA Seq", "GSM2813981", null, "1", "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed and fragmented. The three prime fragments were reverse transcribed and prepared for sequencing Libraries were prepared as described in Zilionis et al. 2017 Nature Protocols PMID = 27929523", "GEO Accession:GSM2813981", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options: appendBCtoName", "DEW155_Lane1.sorted.fastq.gz", "fastq", 1378927876.0, 24382489.0, "GSM2813981 r1", "0:56.55", "A:382023525;C:266729277;G:288053480;T:442115668;N:5926", 56, null, null, null, 382023525, 266729277, 288053480, 442115668, 5926, "SRX3287411", "SRS2596902", "SRA619743", "GEO", "Harvard University", 1, 0.89076, null, 0.20521, null, 0.78281, null, 0.53104, null, 61, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43850, "SRR6176743", "SRX3287411", "SRS2596902", "SRP120009", "PRJNA414416", "Simultaneous single cell profiling of lineages and cell types in the vertebrate brain", "GSE105010", "Other", "The lineage relationships among the hundreds of cell types generated during development are difficult to reconstruct. A recent method GESTALT used CRISPR\u2013Cas9 barcode editing for large scale lineage tracing but was restricted to early development and did not identify cell types. Here we present scGESTALT which combines the lineage recording capabilities of GESTALT with cell type identification by single cell RNA sequencing. The method relies on an inducible system that enables barcodes to be edited at multiple time points capturing lineage information from later stages of development. Sequencing of 60 000 transcriptomes from the juvenile zebrafish brain identified >100 cell types and marker genes. Using these data we generate lineage trees with hundreds of branches that help uncover restrictions at the level of cell types brain regions and gene expression cascades during differentiation. scGESTALT can be applied to other multicellular organisms to simultaneously characterize molecular identities and lineage histories of thousands of cells during development and disease. Overall design: inDrops libraries of single cell transcriptomes and scGESTALT barcodes and genomic DNA GESTALT libraries", null, "pubmed:29608178", null, "DEW155 mid", "GSM2813981", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW155 mid", "Single cell RNA Sequencing data FASTQ files were processed using the inDrops.py bioinformatics pipeline available at https://github.com/indrops/indrops. Transcriptome libraries were mapped to a zebrafish reference built from a custom GTF file and the zebrafish GRCz10 release 86 genome assembly. Bowtie version1.1.1 was used with parameter \u2013e 200; UMI quantification was used with parameter \u2013u 2 counts were ignored from UMIs split between more than 2 genes. genomic DNA GESTALT and scGESTALT libraries were processed using a custom pipeline available at https://github.com/shendurelab/Cas9FateMapping Genome build: GRCz10 CSV files for transcriptome data were generated using the inDrops pipeline. Each column in the CSV files contains a cell identifier and each row contains expression values for genes. Txt files for genomic DNA GESTALT libraries *allReadCounts contain lineage barcode sequences HMID column for each cell and their proportion in the sequenced libraries. scGESTALT data *GestMaster.txt contains the inDrops cell identifiers CellBarcode and BarcodeKey that were used to match barcodes to transcriptomes. They also contain lineage barcode sequences HMID column for each cell with a corresponding inDrops single cell gene expression profile as well as the the t SNE cluster membership number ClusterIdent column Txt files ending in *stats.txt contain information about the each individually captured UMI or cell per sample. The barcode sequence aligned to a reference unedited sequence mergedRead column mutations at each target site target[X] columns and the edited sequences at each target site sequence[X] columns were used for downstream analysis. inDropsExpMatrix noQ txt file is the gene expression matrix for the full dataset. Columns are individual cells from different batches of whole brains f1 f2 f3 f4 f5 f6 or brain regions fore mid hind . fall.inDrops.Robj is the processed Seurat R object which can be loaded into R and explored.", "zebrafish brain", null, "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed and fragmented. The three prime fragments were reverse transcribed and prepared for sequencing Libraries were prepared as described in Zilionis et al. 2017 Nature Protocols PMID = 27929523", null, "tissue:brain|developmental stage:23 25dpf", "GSM2813981", "GSM2813981: DEW155 mid; Danio rerio; RNA Seq", "GSM2813981", null, "1", "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed and fragmented. The three prime fragments were reverse transcribed and prepared for sequencing Libraries were prepared as described in Zilionis et al. 2017 Nature Protocols PMID = 27929523", "GEO Accession:GSM2813981", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options: appendBCtoName", "DEW155_Lane2.sorted.fastq.gz", "fastq", 978765898.0, 17410876.0, "GSM2813981 r2", "0:56.22", "A:270631925;C:189594639;G:205361829;T:313174989;N:2516", 56, null, null, null, 270631925, 189594639, 205361829, 313174989, 2516, "SRX3287411", "SRS2596902", "SRA619743", "GEO", "Harvard University", 1, 0.89018, null, 0.20474, null, 0.78198, null, 0.5019, null, 46, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43851, "SRR6176740", "SRX3287410", "SRS2596884", "SRP120009", "PRJNA414416", "Simultaneous single cell profiling of lineages and cell types in the vertebrate brain", "GSE105010", "Other", "The lineage relationships among the hundreds of cell types generated during development are difficult to reconstruct. A recent method GESTALT used CRISPR\u2013Cas9 barcode editing for large scale lineage tracing but was restricted to early development and did not identify cell types. Here we present scGESTALT which combines the lineage recording capabilities of GESTALT with cell type identification by single cell RNA sequencing. The method relies on an inducible system that enables barcodes to be edited at multiple time points capturing lineage information from later stages of development. Sequencing of 60 000 transcriptomes from the juvenile zebrafish brain identified >100 cell types and marker genes. Using these data we generate lineage trees with hundreds of branches that help uncover restrictions at the level of cell types brain regions and gene expression cascades during differentiation. scGESTALT can be applied to other multicellular organisms to simultaneously characterize molecular identities and lineage histories of thousands of cells during development and disease. Overall design: inDrops libraries of single cell transcriptomes and scGESTALT barcodes and genomic DNA GESTALT libraries", null, "pubmed:29608178", null, "DEW154 hind", "GSM2813980", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW154 hind", "Single cell RNA Sequencing data FASTQ files were processed using the inDrops.py bioinformatics pipeline available at https://github.com/indrops/indrops. Transcriptome libraries were mapped to a zebrafish reference built from a custom GTF file and the zebrafish GRCz10 release 86 genome assembly. Bowtie version1.1.1 was used with parameter \u2013e 200; UMI quantification was used with parameter \u2013u 2 counts were ignored from UMIs split between more than 2 genes. genomic DNA GESTALT and scGESTALT libraries were processed using a custom pipeline available at https://github.com/shendurelab/Cas9FateMapping Genome build: GRCz10 CSV files for transcriptome data were generated using the inDrops pipeline. Each column in the CSV files contains a cell identifier and each row contains expression values for genes. Txt files for genomic DNA GESTALT libraries *allReadCounts contain lineage barcode sequences HMID column for each cell and their proportion in the sequenced libraries. scGESTALT data *GestMaster.txt contains the inDrops cell identifiers CellBarcode and BarcodeKey that were used to match barcodes to transcriptomes. They also contain lineage barcode sequences HMID column for each cell with a corresponding inDrops single cell gene expression profile as well as the the t SNE cluster membership number ClusterIdent column Txt files ending in *stats.txt contain information about the each individually captured UMI or cell per sample. The barcode sequence aligned to a reference unedited sequence mergedRead column mutations at each target site target[X] columns and the edited sequences at each target site sequence[X] columns were used for downstream analysis. inDropsExpMatrix noQ txt file is the gene expression matrix for the full dataset. Columns are individual cells from different batches of whole brains f1 f2 f3 f4 f5 f6 or brain regions fore mid hind . fall.inDrops.Robj is the processed Seurat R object which can be loaded into R and explored.", "zebrafish brain", null, "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed and fragmented. The three prime fragments were reverse transcribed and prepared for sequencing Libraries were prepared as described in Zilionis et al. 2017 Nature Protocols PMID = 27929523", null, "tissue:brain|developmental stage:23 25dpf", "GSM2813980", "GSM2813980: DEW154 hind; Danio rerio; RNA Seq", "GSM2813980", null, "1", "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed and fragmented. The three prime fragments were reverse transcribed and prepared for sequencing Libraries were prepared as described in Zilionis et al. 2017 Nature Protocols PMID = 27929523", "GEO Accession:GSM2813980", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options: appendBCtoName", "DEW154_Lane1.sorted.fastq.gz", "fastq", 191741626.0, 3624944.0, "GSM2813980 r1", "0:52.90", "A:54153798;C:35006191;G:40959293;T:61621602;N:742", 52, null, null, null, 54153798, 35006191, 40959293, 61621602, 742, "SRX3287410", "SRS2596884", "SRA619743", "GEO", "Harvard University", 1, 0.86682, null, 0.2127, null, 0.79695, null, 0.52028, null, 61, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43852, "SRR6176741", "SRX3287410", "SRS2596884", "SRP120009", "PRJNA414416", "Simultaneous single cell profiling of lineages and cell types in the vertebrate brain", "GSE105010", "Other", "The lineage relationships among the hundreds of cell types generated during development are difficult to reconstruct. A recent method GESTALT used CRISPR\u2013Cas9 barcode editing for large scale lineage tracing but was restricted to early development and did not identify cell types. Here we present scGESTALT which combines the lineage recording capabilities of GESTALT with cell type identification by single cell RNA sequencing. The method relies on an inducible system that enables barcodes to be edited at multiple time points capturing lineage information from later stages of development. Sequencing of 60 000 transcriptomes from the juvenile zebrafish brain identified >100 cell types and marker genes. Using these data we generate lineage trees with hundreds of branches that help uncover restrictions at the level of cell types brain regions and gene expression cascades during differentiation. scGESTALT can be applied to other multicellular organisms to simultaneously characterize molecular identities and lineage histories of thousands of cells during development and disease. Overall design: inDrops libraries of single cell transcriptomes and scGESTALT barcodes and genomic DNA GESTALT libraries", null, "pubmed:29608178", null, "DEW154 hind", "GSM2813980", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW154 hind", "Single cell RNA Sequencing data FASTQ files were processed using the inDrops.py bioinformatics pipeline available at https://github.com/indrops/indrops. Transcriptome libraries were mapped to a zebrafish reference built from a custom GTF file and the zebrafish GRCz10 release 86 genome assembly. Bowtie version1.1.1 was used with parameter \u2013e 200; UMI quantification was used with parameter \u2013u 2 counts were ignored from UMIs split between more than 2 genes. genomic DNA GESTALT and scGESTALT libraries were processed using a custom pipeline available at https://github.com/shendurelab/Cas9FateMapping Genome build: GRCz10 CSV files for transcriptome data were generated using the inDrops pipeline. Each column in the CSV files contains a cell identifier and each row contains expression values for genes. Txt files for genomic DNA GESTALT libraries *allReadCounts contain lineage barcode sequences HMID column for each cell and their proportion in the sequenced libraries. scGESTALT data *GestMaster.txt contains the inDrops cell identifiers CellBarcode and BarcodeKey that were used to match barcodes to transcriptomes. They also contain lineage barcode sequences HMID column for each cell with a corresponding inDrops single cell gene expression profile as well as the the t SNE cluster membership number ClusterIdent column Txt files ending in *stats.txt contain information about the each individually captured UMI or cell per sample. The barcode sequence aligned to a reference unedited sequence mergedRead column mutations at each target site target[X] columns and the edited sequences at each target site sequence[X] columns were used for downstream analysis. inDropsExpMatrix noQ txt file is the gene expression matrix for the full dataset. Columns are individual cells from different batches of whole brains f1 f2 f3 f4 f5 f6 or brain regions fore mid hind . fall.inDrops.Robj is the processed Seurat R object which can be loaded into R and explored.", "zebrafish brain", null, "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed and fragmented. The three prime fragments were reverse transcribed and prepared for sequencing Libraries were prepared as described in Zilionis et al. 2017 Nature Protocols PMID = 27929523", null, "tissue:brain|developmental stage:23 25dpf", "GSM2813980", "GSM2813980: DEW154 hind; Danio rerio; RNA Seq", "GSM2813980", null, "1", "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed and fragmented. The three prime fragments were reverse transcribed and prepared for sequencing Libraries were prepared as described in Zilionis et al. 2017 Nature Protocols PMID = 27929523", "GEO Accession:GSM2813980", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options: appendBCtoName", "DEW154_Lane2.sorted.fastq.gz", "fastq", 174428391.0, 3464500.0, "GSM2813980 r2", "0:50.35", "A:49679029;C:31732468;G:36877474;T:56139060;N:360", 50, null, null, null, 49679029, 31732468, 36877474, 56139060, 360, "SRX3287410", "SRS2596884", "SRA619743", "GEO", "Harvard University", 1, 0.85562, null, 0.21627, null, 0.79839, null, 0.52737, null, 61, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43853, "SRR6176738", "SRX3287409", "SRS2596883", "SRP120009", "PRJNA414416", "Simultaneous single cell profiling of lineages and cell types in the vertebrate brain", "GSE105010", "Other", "The lineage relationships among the hundreds of cell types generated during development are difficult to reconstruct. A recent method GESTALT used CRISPR\u2013Cas9 barcode editing for large scale lineage tracing but was restricted to early development and did not identify cell types. Here we present scGESTALT which combines the lineage recording capabilities of GESTALT with cell type identification by single cell RNA sequencing. The method relies on an inducible system that enables barcodes to be edited at multiple time points capturing lineage information from later stages of development. Sequencing of 60 000 transcriptomes from the juvenile zebrafish brain identified >100 cell types and marker genes. Using these data we generate lineage trees with hundreds of branches that help uncover restrictions at the level of cell types brain regions and gene expression cascades during differentiation. scGESTALT can be applied to other multicellular organisms to simultaneously characterize molecular identities and lineage histories of thousands of cells during development and disease. Overall design: inDrops libraries of single cell transcriptomes and scGESTALT barcodes and genomic DNA GESTALT libraries", null, "pubmed:29608178", null, "DEW153 hind", "GSM2813979", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW153 hind", "Single cell RNA Sequencing data FASTQ files were processed using the inDrops.py bioinformatics pipeline available at https://github.com/indrops/indrops. Transcriptome libraries were mapped to a zebrafish reference built from a custom GTF file and the zebrafish GRCz10 release 86 genome assembly. Bowtie version1.1.1 was used with parameter \u2013e 200; UMI quantification was used with parameter \u2013u 2 counts were ignored from UMIs split between more than 2 genes. genomic DNA GESTALT and scGESTALT libraries were processed using a custom pipeline available at https://github.com/shendurelab/Cas9FateMapping Genome build: GRCz10 CSV files for transcriptome data were generated using the inDrops pipeline. Each column in the CSV files contains a cell identifier and each row contains expression values for genes. Txt files for genomic DNA GESTALT libraries *allReadCounts contain lineage barcode sequences HMID column for each cell and their proportion in the sequenced libraries. scGESTALT data *GestMaster.txt contains the inDrops cell identifiers CellBarcode and BarcodeKey that were used to match barcodes to transcriptomes. They also contain lineage barcode sequences HMID column for each cell with a corresponding inDrops single cell gene expression profile as well as the the t SNE cluster membership number ClusterIdent column Txt files ending in *stats.txt contain information about the each individually captured UMI or cell per sample. The barcode sequence aligned to a reference unedited sequence mergedRead column mutations at each target site target[X] columns and the edited sequences at each target site sequence[X] columns were used for downstream analysis. inDropsExpMatrix noQ txt file is the gene expression matrix for the full dataset. Columns are individual cells from different batches of whole brains f1 f2 f3 f4 f5 f6 or brain regions fore mid hind . fall.inDrops.Robj is the processed Seurat R object which can be loaded into R and explored.", "zebrafish brain", null, "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed and fragmented. The three prime fragments were reverse transcribed and prepared for sequencing Libraries were prepared as described in Zilionis et al. 2017 Nature Protocols PMID = 27929523", null, "tissue:brain|developmental stage:23 25dpf", "GSM2813979", "GSM2813979: DEW153 hind; Danio rerio; RNA Seq", "GSM2813979", null, "1", "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed and fragmented. The three prime fragments were reverse transcribed and prepared for sequencing Libraries were prepared as described in Zilionis et al. 2017 Nature Protocols PMID = 27929523", "GEO Accession:GSM2813979", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options: appendBCtoName", "DEW153_Lane1.sorted.fastq.gz", "fastq", 2206528171.0, 38115978.0, "GSM2813979 r1", "0:57.89", "A:607831752;C:435134646;G:462618869;T:700932389;N:10515", 57, null, null, null, 607831752, 435134646, 462618869, 700932389, 10515, "SRX3287409", "SRS2596883", "SRA619743", "GEO", "Harvard University", 1, 0.89527, null, 0.20046, null, 0.78259, null, 0.50876, null, 60, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43854, "SRR6176739", "SRX3287409", "SRS2596883", "SRP120009", "PRJNA414416", "Simultaneous single cell profiling of lineages and cell types in the vertebrate brain", "GSE105010", "Other", "The lineage relationships among the hundreds of cell types generated during development are difficult to reconstruct. A recent method GESTALT used CRISPR\u2013Cas9 barcode editing for large scale lineage tracing but was restricted to early development and did not identify cell types. Here we present scGESTALT which combines the lineage recording capabilities of GESTALT with cell type identification by single cell RNA sequencing. The method relies on an inducible system that enables barcodes to be edited at multiple time points capturing lineage information from later stages of development. Sequencing of 60 000 transcriptomes from the juvenile zebrafish brain identified >100 cell types and marker genes. Using these data we generate lineage trees with hundreds of branches that help uncover restrictions at the level of cell types brain regions and gene expression cascades during differentiation. scGESTALT can be applied to other multicellular organisms to simultaneously characterize molecular identities and lineage histories of thousands of cells during development and disease. Overall design: inDrops libraries of single cell transcriptomes and scGESTALT barcodes and genomic DNA GESTALT libraries", null, "pubmed:29608178", null, "DEW153 hind", "GSM2813979", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW153 hind", "Single cell RNA Sequencing data FASTQ files were processed using the inDrops.py bioinformatics pipeline available at https://github.com/indrops/indrops. Transcriptome libraries were mapped to a zebrafish reference built from a custom GTF file and the zebrafish GRCz10 release 86 genome assembly. Bowtie version1.1.1 was used with parameter \u2013e 200; UMI quantification was used with parameter \u2013u 2 counts were ignored from UMIs split between more than 2 genes. genomic DNA GESTALT and scGESTALT libraries were processed using a custom pipeline available at https://github.com/shendurelab/Cas9FateMapping Genome build: GRCz10 CSV files for transcriptome data were generated using the inDrops pipeline. Each column in the CSV files contains a cell identifier and each row contains expression values for genes. Txt files for genomic DNA GESTALT libraries *allReadCounts contain lineage barcode sequences HMID column for each cell and their proportion in the sequenced libraries. scGESTALT data *GestMaster.txt contains the inDrops cell identifiers CellBarcode and BarcodeKey that were used to match barcodes to transcriptomes. They also contain lineage barcode sequences HMID column for each cell with a corresponding inDrops single cell gene expression profile as well as the the t SNE cluster membership number ClusterIdent column Txt files ending in *stats.txt contain information about the each individually captured UMI or cell per sample. The barcode sequence aligned to a reference unedited sequence mergedRead column mutations at each target site target[X] columns and the edited sequences at each target site sequence[X] columns were used for downstream analysis. inDropsExpMatrix noQ txt file is the gene expression matrix for the full dataset. Columns are individual cells from different batches of whole brains f1 f2 f3 f4 f5 f6 or brain regions fore mid hind . fall.inDrops.Robj is the processed Seurat R object which can be loaded into R and explored.", "zebrafish brain", null, "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed and fragmented. The three prime fragments were reverse transcribed and prepared for sequencing Libraries were prepared as described in Zilionis et al. 2017 Nature Protocols PMID = 27929523", null, "tissue:brain|developmental stage:23 25dpf", "GSM2813979", "GSM2813979: DEW153 hind; Danio rerio; RNA Seq", "GSM2813979", null, "1", "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed and fragmented. The three prime fragments were reverse transcribed and prepared for sequencing Libraries were prepared as described in Zilionis et al. 2017 Nature Protocols PMID = 27929523", "GEO Accession:GSM2813979", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options: appendBCtoName", "DEW153_Lane2.sorted.fastq.gz", "fastq", 1450777748.0, 25293610.0, "GSM2813979 r2", "0:57.36", "A:398797215;C:285454365;G:305822323;T:460699598;N:4247", 57, null, null, null, 398797215, 285454365, 305822323, 460699598, 4247, "SRX3287409", "SRS2596883", "SRA619743", "GEO", "Harvard University", 1, 0.89512, null, 0.20047, null, 0.78222, null, 0.53369, null, 60, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43855, "SRR6176736", "SRX3287408", "SRS2596882", "SRP120009", "PRJNA414416", "Simultaneous single cell profiling of lineages and cell types in the vertebrate brain", "GSE105010", "Other", "The lineage relationships among the hundreds of cell types generated during development are difficult to reconstruct. A recent method GESTALT used CRISPR\u2013Cas9 barcode editing for large scale lineage tracing but was restricted to early development and did not identify cell types. Here we present scGESTALT which combines the lineage recording capabilities of GESTALT with cell type identification by single cell RNA sequencing. The method relies on an inducible system that enables barcodes to be edited at multiple time points capturing lineage information from later stages of development. Sequencing of 60 000 transcriptomes from the juvenile zebrafish brain identified >100 cell types and marker genes. Using these data we generate lineage trees with hundreds of branches that help uncover restrictions at the level of cell types brain regions and gene expression cascades during differentiation. scGESTALT can be applied to other multicellular organisms to simultaneously characterize molecular identities and lineage histories of thousands of cells during development and disease. Overall design: inDrops libraries of single cell transcriptomes and scGESTALT barcodes and genomic DNA GESTALT libraries", null, "pubmed:29608178", null, "DEW152 hind", "GSM2813978", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW152 hind", "Single cell RNA Sequencing data FASTQ files were processed using the inDrops.py bioinformatics pipeline available at https://github.com/indrops/indrops. Transcriptome libraries were mapped to a zebrafish reference built from a custom GTF file and the zebrafish GRCz10 release 86 genome assembly. Bowtie version1.1.1 was used with parameter \u2013e 200; UMI quantification was used with parameter \u2013u 2 counts were ignored from UMIs split between more than 2 genes. genomic DNA GESTALT and scGESTALT libraries were processed using a custom pipeline available at https://github.com/shendurelab/Cas9FateMapping Genome build: GRCz10 CSV files for transcriptome data were generated using the inDrops pipeline. Each column in the CSV files contains a cell identifier and each row contains expression values for genes. Txt files for genomic DNA GESTALT libraries *allReadCounts contain lineage barcode sequences HMID column for each cell and their proportion in the sequenced libraries. scGESTALT data *GestMaster.txt contains the inDrops cell identifiers CellBarcode and BarcodeKey that were used to match barcodes to transcriptomes. They also contain lineage barcode sequences HMID column for each cell with a corresponding inDrops single cell gene expression profile as well as the the t SNE cluster membership number ClusterIdent column Txt files ending in *stats.txt contain information about the each individually captured UMI or cell per sample. The barcode sequence aligned to a reference unedited sequence mergedRead column mutations at each target site target[X] columns and the edited sequences at each target site sequence[X] columns were used for downstream analysis. inDropsExpMatrix noQ txt file is the gene expression matrix for the full dataset. Columns are individual cells from different batches of whole brains f1 f2 f3 f4 f5 f6 or brain regions fore mid hind . fall.inDrops.Robj is the processed Seurat R object which can be loaded into R and explored.", "zebrafish brain", null, "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed and fragmented. The three prime fragments were reverse transcribed and prepared for sequencing Libraries were prepared as described in Zilionis et al. 2017 Nature Protocols PMID = 27929523", null, "tissue:brain|developmental stage:23 25dpf", "GSM2813978", "GSM2813978: DEW152 hind; Danio rerio; RNA Seq", "GSM2813978", null, "1", "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed and fragmented. The three prime fragments were reverse transcribed and prepared for sequencing Libraries were prepared as described in Zilionis et al. 2017 Nature Protocols PMID = 27929523", "GEO Accession:GSM2813978", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options: appendBCtoName", "DEW152_Lane1.sorted.fastq.gz", "fastq", 2005997266.0, 34574429.0, "GSM2813978 r1", "0:58.02", "A:552258277;C:393949059;G:416248897;T:643531466;N:9567", 58, null, null, null, 552258277, 393949059, 416248897, 643531466, 9567, "SRX3287408", "SRS2596882", "SRA619743", "GEO", "Harvard University", 1, 0.89818, null, 0.20396, null, 0.78001, null, 0.5176, null, 61, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43856, "SRR6176737", "SRX3287408", "SRS2596882", "SRP120009", "PRJNA414416", "Simultaneous single cell profiling of lineages and cell types in the vertebrate brain", "GSE105010", "Other", "The lineage relationships among the hundreds of cell types generated during development are difficult to reconstruct. A recent method GESTALT used CRISPR\u2013Cas9 barcode editing for large scale lineage tracing but was restricted to early development and did not identify cell types. Here we present scGESTALT which combines the lineage recording capabilities of GESTALT with cell type identification by single cell RNA sequencing. The method relies on an inducible system that enables barcodes to be edited at multiple time points capturing lineage information from later stages of development. Sequencing of 60 000 transcriptomes from the juvenile zebrafish brain identified >100 cell types and marker genes. Using these data we generate lineage trees with hundreds of branches that help uncover restrictions at the level of cell types brain regions and gene expression cascades during differentiation. scGESTALT can be applied to other multicellular organisms to simultaneously characterize molecular identities and lineage histories of thousands of cells during development and disease. Overall design: inDrops libraries of single cell transcriptomes and scGESTALT barcodes and genomic DNA GESTALT libraries", null, "pubmed:29608178", null, "DEW152 hind", "GSM2813978", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW152 hind", "Single cell RNA Sequencing data FASTQ files were processed using the inDrops.py bioinformatics pipeline available at https://github.com/indrops/indrops. Transcriptome libraries were mapped to a zebrafish reference built from a custom GTF file and the zebrafish GRCz10 release 86 genome assembly. Bowtie version1.1.1 was used with parameter \u2013e 200; UMI quantification was used with parameter \u2013u 2 counts were ignored from UMIs split between more than 2 genes. genomic DNA GESTALT and scGESTALT libraries were processed using a custom pipeline available at https://github.com/shendurelab/Cas9FateMapping Genome build: GRCz10 CSV files for transcriptome data were generated using the inDrops pipeline. Each column in the CSV files contains a cell identifier and each row contains expression values for genes. Txt files for genomic DNA GESTALT libraries *allReadCounts contain lineage barcode sequences HMID column for each cell and their proportion in the sequenced libraries. scGESTALT data *GestMaster.txt contains the inDrops cell identifiers CellBarcode and BarcodeKey that were used to match barcodes to transcriptomes. They also contain lineage barcode sequences HMID column for each cell with a corresponding inDrops single cell gene expression profile as well as the the t SNE cluster membership number ClusterIdent column Txt files ending in *stats.txt contain information about the each individually captured UMI or cell per sample. The barcode sequence aligned to a reference unedited sequence mergedRead column mutations at each target site target[X] columns and the edited sequences at each target site sequence[X] columns were used for downstream analysis. inDropsExpMatrix noQ txt file is the gene expression matrix for the full dataset. Columns are individual cells from different batches of whole brains f1 f2 f3 f4 f5 f6 or brain regions fore mid hind . fall.inDrops.Robj is the processed Seurat R object which can be loaded into R and explored.", "zebrafish brain", null, "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed and fragmented. The three prime fragments were reverse transcribed and prepared for sequencing Libraries were prepared as described in Zilionis et al. 2017 Nature Protocols PMID = 27929523", null, "tissue:brain|developmental stage:23 25dpf", "GSM2813978", "GSM2813978: DEW152 hind; Danio rerio; RNA Seq", "GSM2813978", null, "1", "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed and fragmented. The three prime fragments were reverse transcribed and prepared for sequencing Libraries were prepared as described in Zilionis et al. 2017 Nature Protocols PMID = 27929523", "GEO Accession:GSM2813978", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options: appendBCtoName", "DEW152_Lane2.sorted.fastq.gz", "fastq", 1306624373.0, 22721229.0, "GSM2813978 r2", "0:57.51", "A:358953147;C:256408571;G:272527726;T:418731243;N:3686", 57, null, null, null, 358953147, 256408571, 272527726, 418731243, 3686, "SRX3287408", "SRS2596882", "SRA619743", "GEO", "Harvard University", 1, 0.89769, null, 0.20479, null, 0.78204, null, 0.51518, null, 61, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43857, "SRR6176734", "SRX3287407", "SRS2596880", "SRP120009", "PRJNA414416", "Simultaneous single cell profiling of lineages and cell types in the vertebrate brain", "GSE105010", "Other", "The lineage relationships among the hundreds of cell types generated during development are difficult to reconstruct. A recent method GESTALT used CRISPR\u2013Cas9 barcode editing for large scale lineage tracing but was restricted to early development and did not identify cell types. Here we present scGESTALT which combines the lineage recording capabilities of GESTALT with cell type identification by single cell RNA sequencing. The method relies on an inducible system that enables barcodes to be edited at multiple time points capturing lineage information from later stages of development. Sequencing of 60 000 transcriptomes from the juvenile zebrafish brain identified >100 cell types and marker genes. Using these data we generate lineage trees with hundreds of branches that help uncover restrictions at the level of cell types brain regions and gene expression cascades during differentiation. scGESTALT can be applied to other multicellular organisms to simultaneously characterize molecular identities and lineage histories of thousands of cells during development and disease. Overall design: inDrops libraries of single cell transcriptomes and scGESTALT barcodes and genomic DNA GESTALT libraries", null, "pubmed:29608178", null, "DEW151 fore", "GSM2813977", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW151 fore", "Single cell RNA Sequencing data FASTQ files were processed using the inDrops.py bioinformatics pipeline available at https://github.com/indrops/indrops. Transcriptome libraries were mapped to a zebrafish reference built from a custom GTF file and the zebrafish GRCz10 release 86 genome assembly. Bowtie version1.1.1 was used with parameter \u2013e 200; UMI quantification was used with parameter \u2013u 2 counts were ignored from UMIs split between more than 2 genes. genomic DNA GESTALT and scGESTALT libraries were processed using a custom pipeline available at https://github.com/shendurelab/Cas9FateMapping Genome build: GRCz10 CSV files for transcriptome data were generated using the inDrops pipeline. Each column in the CSV files contains a cell identifier and each row contains expression values for genes. Txt files for genomic DNA GESTALT libraries *allReadCounts contain lineage barcode sequences HMID column for each cell and their proportion in the sequenced libraries. scGESTALT data *GestMaster.txt contains the inDrops cell identifiers CellBarcode and BarcodeKey that were used to match barcodes to transcriptomes. They also contain lineage barcode sequences HMID column for each cell with a corresponding inDrops single cell gene expression profile as well as the the t SNE cluster membership number ClusterIdent column Txt files ending in *stats.txt contain information about the each individually captured UMI or cell per sample. The barcode sequence aligned to a reference unedited sequence mergedRead column mutations at each target site target[X] columns and the edited sequences at each target site sequence[X] columns were used for downstream analysis. inDropsExpMatrix noQ txt file is the gene expression matrix for the full dataset. Columns are individual cells from different batches of whole brains f1 f2 f3 f4 f5 f6 or brain regions fore mid hind . fall.inDrops.Robj is the processed Seurat R object which can be loaded into R and explored.", "zebrafish brain", null, "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed and fragmented. The three prime fragments were reverse transcribed and prepared for sequencing Libraries were prepared as described in Zilionis et al. 2017 Nature Protocols PMID = 27929523", null, "tissue:brain|developmental stage:23 25dpf", "GSM2813977", "GSM2813977: DEW151 fore; Danio rerio; RNA Seq", "GSM2813977", null, "1", "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed and fragmented. The three prime fragments were reverse transcribed and prepared for sequencing Libraries were prepared as described in Zilionis et al. 2017 Nature Protocols PMID = 27929523", "GEO Accession:GSM2813977", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options: appendBCtoName", "DEW151_Lane1.sorted.fastq.gz", "fastq", 1781584043.0, 30810377.0, "GSM2813977 r1", "0:57.82", "A:494848942;C:336194488;G:366298578;T:584233757;N:8278", 57, null, null, null, 494848942, 336194488, 366298578, 584233757, 8278, "SRX3287407", "SRS2596880", "SRA619743", "GEO", "Harvard University", 1, 0.88247, null, 0.27771, null, 0.78311, null, 0.52819, null, 61, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43858, "SRR6176735", "SRX3287407", "SRS2596880", "SRP120009", "PRJNA414416", "Simultaneous single cell profiling of lineages and cell types in the vertebrate brain", "GSE105010", "Other", "The lineage relationships among the hundreds of cell types generated during development are difficult to reconstruct. A recent method GESTALT used CRISPR\u2013Cas9 barcode editing for large scale lineage tracing but was restricted to early development and did not identify cell types. Here we present scGESTALT which combines the lineage recording capabilities of GESTALT with cell type identification by single cell RNA sequencing. The method relies on an inducible system that enables barcodes to be edited at multiple time points capturing lineage information from later stages of development. Sequencing of 60 000 transcriptomes from the juvenile zebrafish brain identified >100 cell types and marker genes. Using these data we generate lineage trees with hundreds of branches that help uncover restrictions at the level of cell types brain regions and gene expression cascades during differentiation. scGESTALT can be applied to other multicellular organisms to simultaneously characterize molecular identities and lineage histories of thousands of cells during development and disease. Overall design: inDrops libraries of single cell transcriptomes and scGESTALT barcodes and genomic DNA GESTALT libraries", null, "pubmed:29608178", null, "DEW151 fore", "GSM2813977", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW151 fore", "Single cell RNA Sequencing data FASTQ files were processed using the inDrops.py bioinformatics pipeline available at https://github.com/indrops/indrops. Transcriptome libraries were mapped to a zebrafish reference built from a custom GTF file and the zebrafish GRCz10 release 86 genome assembly. Bowtie version1.1.1 was used with parameter \u2013e 200; UMI quantification was used with parameter \u2013u 2 counts were ignored from UMIs split between more than 2 genes. genomic DNA GESTALT and scGESTALT libraries were processed using a custom pipeline available at https://github.com/shendurelab/Cas9FateMapping Genome build: GRCz10 CSV files for transcriptome data were generated using the inDrops pipeline. Each column in the CSV files contains a cell identifier and each row contains expression values for genes. Txt files for genomic DNA GESTALT libraries *allReadCounts contain lineage barcode sequences HMID column for each cell and their proportion in the sequenced libraries. scGESTALT data *GestMaster.txt contains the inDrops cell identifiers CellBarcode and BarcodeKey that were used to match barcodes to transcriptomes. They also contain lineage barcode sequences HMID column for each cell with a corresponding inDrops single cell gene expression profile as well as the the t SNE cluster membership number ClusterIdent column Txt files ending in *stats.txt contain information about the each individually captured UMI or cell per sample. The barcode sequence aligned to a reference unedited sequence mergedRead column mutations at each target site target[X] columns and the edited sequences at each target site sequence[X] columns were used for downstream analysis. inDropsExpMatrix noQ txt file is the gene expression matrix for the full dataset. Columns are individual cells from different batches of whole brains f1 f2 f3 f4 f5 f6 or brain regions fore mid hind . fall.inDrops.Robj is the processed Seurat R object which can be loaded into R and explored.", "zebrafish brain", null, "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed and fragmented. The three prime fragments were reverse transcribed and prepared for sequencing Libraries were prepared as described in Zilionis et al. 2017 Nature Protocols PMID = 27929523", null, "tissue:brain|developmental stage:23 25dpf", "GSM2813977", "GSM2813977: DEW151 fore; Danio rerio; RNA Seq", "GSM2813977", null, "1", "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed and fragmented. The three prime fragments were reverse transcribed and prepared for sequencing Libraries were prepared as described in Zilionis et al. 2017 Nature Protocols PMID = 27929523", "GEO Accession:GSM2813977", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options: appendBCtoName", "DEW151_Lane2.sorted.fastq.gz", "fastq", 1157526748.0, 20213966.0, "GSM2813977 r2", "0:57.26", "A:320886308;C:218034938;G:239168032;T:379434373;N:3097", 57, null, null, null, 320886308, 218034938, 239168032, 379434373, 3097, "SRX3287407", "SRS2596880", "SRA619743", "GEO", "Harvard University", 1, 0.88331, null, 0.27805, null, 0.78599, null, 0.52469, null, 60, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43859, "SRR6176732", "SRX3287406", "SRS2596881", "SRP120009", "PRJNA414416", "Simultaneous single cell profiling of lineages and cell types in the vertebrate brain", "GSE105010", "Other", "The lineage relationships among the hundreds of cell types generated during development are difficult to reconstruct. A recent method GESTALT used CRISPR\u2013Cas9 barcode editing for large scale lineage tracing but was restricted to early development and did not identify cell types. Here we present scGESTALT which combines the lineage recording capabilities of GESTALT with cell type identification by single cell RNA sequencing. The method relies on an inducible system that enables barcodes to be edited at multiple time points capturing lineage information from later stages of development. Sequencing of 60 000 transcriptomes from the juvenile zebrafish brain identified >100 cell types and marker genes. Using these data we generate lineage trees with hundreds of branches that help uncover restrictions at the level of cell types brain regions and gene expression cascades during differentiation. scGESTALT can be applied to other multicellular organisms to simultaneously characterize molecular identities and lineage histories of thousands of cells during development and disease. Overall design: inDrops libraries of single cell transcriptomes and scGESTALT barcodes and genomic DNA GESTALT libraries", null, "pubmed:29608178", null, "DEW150 fore", "GSM2813976", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW150 fore", "Single cell RNA Sequencing data FASTQ files were processed using the inDrops.py bioinformatics pipeline available at https://github.com/indrops/indrops. Transcriptome libraries were mapped to a zebrafish reference built from a custom GTF file and the zebrafish GRCz10 release 86 genome assembly. Bowtie version1.1.1 was used with parameter \u2013e 200; UMI quantification was used with parameter \u2013u 2 counts were ignored from UMIs split between more than 2 genes. genomic DNA GESTALT and scGESTALT libraries were processed using a custom pipeline available at https://github.com/shendurelab/Cas9FateMapping Genome build: GRCz10 CSV files for transcriptome data were generated using the inDrops pipeline. Each column in the CSV files contains a cell identifier and each row contains expression values for genes. Txt files for genomic DNA GESTALT libraries *allReadCounts contain lineage barcode sequences HMID column for each cell and their proportion in the sequenced libraries. scGESTALT data *GestMaster.txt contains the inDrops cell identifiers CellBarcode and BarcodeKey that were used to match barcodes to transcriptomes. They also contain lineage barcode sequences HMID column for each cell with a corresponding inDrops single cell gene expression profile as well as the the t SNE cluster membership number ClusterIdent column Txt files ending in *stats.txt contain information about the each individually captured UMI or cell per sample. The barcode sequence aligned to a reference unedited sequence mergedRead column mutations at each target site target[X] columns and the edited sequences at each target site sequence[X] columns were used for downstream analysis. inDropsExpMatrix noQ txt file is the gene expression matrix for the full dataset. Columns are individual cells from different batches of whole brains f1 f2 f3 f4 f5 f6 or brain regions fore mid hind . fall.inDrops.Robj is the processed Seurat R object which can be loaded into R and explored.", "zebrafish brain", null, "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed and fragmented. The three prime fragments were reverse transcribed and prepared for sequencing Libraries were prepared as described in Zilionis et al. 2017 Nature Protocols PMID = 27929523", null, "tissue:brain|developmental stage:23 25dpf", "GSM2813976", "GSM2813976: DEW150 fore; Danio rerio; RNA Seq", "GSM2813976", null, "1", "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed and fragmented. The three prime fragments were reverse transcribed and prepared for sequencing Libraries were prepared as described in Zilionis et al. 2017 Nature Protocols PMID = 27929523", "GEO Accession:GSM2813976", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options: appendBCtoName", "DEW150_Lane1.sorted.fastq.gz", "fastq", 2032276612.0, 35123226.0, "GSM2813976 r1", "0:57.86", "A:563752104;C:383031134;G:429113539;T:656370389;N:9446", 57, null, null, null, 563752104, 383031134, 429113539, 656370389, 9446, "SRX3287406", "SRS2596881", "SRA619743", "GEO", "Harvard University", 1, 0.8788, null, 0.27566, null, 0.78995, null, 0.521, null, 61, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43860, "SRR6176733", "SRX3287406", "SRS2596881", "SRP120009", "PRJNA414416", "Simultaneous single cell profiling of lineages and cell types in the vertebrate brain", "GSE105010", "Other", "The lineage relationships among the hundreds of cell types generated during development are difficult to reconstruct. A recent method GESTALT used CRISPR\u2013Cas9 barcode editing for large scale lineage tracing but was restricted to early development and did not identify cell types. Here we present scGESTALT which combines the lineage recording capabilities of GESTALT with cell type identification by single cell RNA sequencing. The method relies on an inducible system that enables barcodes to be edited at multiple time points capturing lineage information from later stages of development. Sequencing of 60 000 transcriptomes from the juvenile zebrafish brain identified >100 cell types and marker genes. Using these data we generate lineage trees with hundreds of branches that help uncover restrictions at the level of cell types brain regions and gene expression cascades during differentiation. scGESTALT can be applied to other multicellular organisms to simultaneously characterize molecular identities and lineage histories of thousands of cells during development and disease. Overall design: inDrops libraries of single cell transcriptomes and scGESTALT barcodes and genomic DNA GESTALT libraries", null, "pubmed:29608178", null, "DEW150 fore", "GSM2813976", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW150 fore", "Single cell RNA Sequencing data FASTQ files were processed using the inDrops.py bioinformatics pipeline available at https://github.com/indrops/indrops. Transcriptome libraries were mapped to a zebrafish reference built from a custom GTF file and the zebrafish GRCz10 release 86 genome assembly. Bowtie version1.1.1 was used with parameter \u2013e 200; UMI quantification was used with parameter \u2013u 2 counts were ignored from UMIs split between more than 2 genes. genomic DNA GESTALT and scGESTALT libraries were processed using a custom pipeline available at https://github.com/shendurelab/Cas9FateMapping Genome build: GRCz10 CSV files for transcriptome data were generated using the inDrops pipeline. Each column in the CSV files contains a cell identifier and each row contains expression values for genes. Txt files for genomic DNA GESTALT libraries *allReadCounts contain lineage barcode sequences HMID column for each cell and their proportion in the sequenced libraries. scGESTALT data *GestMaster.txt contains the inDrops cell identifiers CellBarcode and BarcodeKey that were used to match barcodes to transcriptomes. They also contain lineage barcode sequences HMID column for each cell with a corresponding inDrops single cell gene expression profile as well as the the t SNE cluster membership number ClusterIdent column Txt files ending in *stats.txt contain information about the each individually captured UMI or cell per sample. The barcode sequence aligned to a reference unedited sequence mergedRead column mutations at each target site target[X] columns and the edited sequences at each target site sequence[X] columns were used for downstream analysis. inDropsExpMatrix noQ txt file is the gene expression matrix for the full dataset. Columns are individual cells from different batches of whole brains f1 f2 f3 f4 f5 f6 or brain regions fore mid hind . fall.inDrops.Robj is the processed Seurat R object which can be loaded into R and explored.", "zebrafish brain", null, "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed and fragmented. The three prime fragments were reverse transcribed and prepared for sequencing Libraries were prepared as described in Zilionis et al. 2017 Nature Protocols PMID = 27929523", null, "tissue:brain|developmental stage:23 25dpf", "GSM2813976", "GSM2813976: DEW150 fore; Danio rerio; RNA Seq", "GSM2813976", null, "1", "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed and fragmented. The three prime fragments were reverse transcribed and prepared for sequencing Libraries were prepared as described in Zilionis et al. 2017 Nature Protocols PMID = 27929523", "GEO Accession:GSM2813976", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options: appendBCtoName", "DEW150_Lane2.sorted.fastq.gz", "fastq", 1315848593.0, 22939860.0, "GSM2813976 r2", "0:57.36", "A:364153526;C:247636153;G:279420005;T:424635138;N:3771", 57, null, null, null, 364153526, 247636153, 279420005, 424635138, 3771, "SRX3287406", "SRS2596881", "SRA619743", "GEO", "Harvard University", 1, 0.87913, null, 0.27404, null, 0.78666, null, 0.52726, null, 61, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43861, "SRR6176730", "SRX3287405", "SRS2596879", "SRP120009", "PRJNA414416", "Simultaneous single cell profiling of lineages and cell types in the vertebrate brain", "GSE105010", "Other", "The lineage relationships among the hundreds of cell types generated during development are difficult to reconstruct. A recent method GESTALT used CRISPR\u2013Cas9 barcode editing for large scale lineage tracing but was restricted to early development and did not identify cell types. Here we present scGESTALT which combines the lineage recording capabilities of GESTALT with cell type identification by single cell RNA sequencing. The method relies on an inducible system that enables barcodes to be edited at multiple time points capturing lineage information from later stages of development. Sequencing of 60 000 transcriptomes from the juvenile zebrafish brain identified >100 cell types and marker genes. Using these data we generate lineage trees with hundreds of branches that help uncover restrictions at the level of cell types brain regions and gene expression cascades during differentiation. scGESTALT can be applied to other multicellular organisms to simultaneously characterize molecular identities and lineage histories of thousands of cells during development and disease. Overall design: inDrops libraries of single cell transcriptomes and scGESTALT barcodes and genomic DNA GESTALT libraries", null, "pubmed:29608178", null, "DEW149 fore", "GSM2813975", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW149 fore", "Single cell RNA Sequencing data FASTQ files were processed using the inDrops.py bioinformatics pipeline available at https://github.com/indrops/indrops. Transcriptome libraries were mapped to a zebrafish reference built from a custom GTF file and the zebrafish GRCz10 release 86 genome assembly. Bowtie version1.1.1 was used with parameter \u2013e 200; UMI quantification was used with parameter \u2013u 2 counts were ignored from UMIs split between more than 2 genes. genomic DNA GESTALT and scGESTALT libraries were processed using a custom pipeline available at https://github.com/shendurelab/Cas9FateMapping Genome build: GRCz10 CSV files for transcriptome data were generated using the inDrops pipeline. Each column in the CSV files contains a cell identifier and each row contains expression values for genes. Txt files for genomic DNA GESTALT libraries *allReadCounts contain lineage barcode sequences HMID column for each cell and their proportion in the sequenced libraries. scGESTALT data *GestMaster.txt contains the inDrops cell identifiers CellBarcode and BarcodeKey that were used to match barcodes to transcriptomes. They also contain lineage barcode sequences HMID column for each cell with a corresponding inDrops single cell gene expression profile as well as the the t SNE cluster membership number ClusterIdent column Txt files ending in *stats.txt contain information about the each individually captured UMI or cell per sample. The barcode sequence aligned to a reference unedited sequence mergedRead column mutations at each target site target[X] columns and the edited sequences at each target site sequence[X] columns were used for downstream analysis. inDropsExpMatrix noQ txt file is the gene expression matrix for the full dataset. Columns are individual cells from different batches of whole brains f1 f2 f3 f4 f5 f6 or brain regions fore mid hind . fall.inDrops.Robj is the processed Seurat R object which can be loaded into R and explored.", "zebrafish brain", null, "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed and fragmented. The three prime fragments were reverse transcribed and prepared for sequencing Libraries were prepared as described in Zilionis et al. 2017 Nature Protocols PMID = 27929523", null, "tissue:brain|developmental stage:23 25dpf", "GSM2813975", "GSM2813975: DEW149 fore; Danio rerio; RNA Seq", "GSM2813975", null, "1", "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed and fragmented. The three prime fragments were reverse transcribed and prepared for sequencing Libraries were prepared as described in Zilionis et al. 2017 Nature Protocols PMID = 27929523", "GEO Accession:GSM2813975", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options: appendBCtoName", "DEW149_Lane1.sorted.fastq.gz", "fastq", 2040984134.0, 35277532.0, "GSM2813975 r1", "0:57.86", "A:567524269;C:385217324;G:426974572;T:661258382;N:9587", 57, null, null, null, 567524269, 385217324, 426974572, 661258382, 9587, "SRX3287405", "SRS2596879", "SRA619743", "GEO", "Harvard University", 1, 0.8811, null, 0.27464, null, 0.78654, null, 0.52635, null, 61, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43862, "SRR6176731", "SRX3287405", "SRS2596879", "SRP120009", "PRJNA414416", "Simultaneous single cell profiling of lineages and cell types in the vertebrate brain", "GSE105010", "Other", "The lineage relationships among the hundreds of cell types generated during development are difficult to reconstruct. A recent method GESTALT used CRISPR\u2013Cas9 barcode editing for large scale lineage tracing but was restricted to early development and did not identify cell types. Here we present scGESTALT which combines the lineage recording capabilities of GESTALT with cell type identification by single cell RNA sequencing. The method relies on an inducible system that enables barcodes to be edited at multiple time points capturing lineage information from later stages of development. Sequencing of 60 000 transcriptomes from the juvenile zebrafish brain identified >100 cell types and marker genes. Using these data we generate lineage trees with hundreds of branches that help uncover restrictions at the level of cell types brain regions and gene expression cascades during differentiation. scGESTALT can be applied to other multicellular organisms to simultaneously characterize molecular identities and lineage histories of thousands of cells during development and disease. Overall design: inDrops libraries of single cell transcriptomes and scGESTALT barcodes and genomic DNA GESTALT libraries", null, "pubmed:29608178", null, "DEW149 fore", "GSM2813975", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW149 fore", "Single cell RNA Sequencing data FASTQ files were processed using the inDrops.py bioinformatics pipeline available at https://github.com/indrops/indrops. Transcriptome libraries were mapped to a zebrafish reference built from a custom GTF file and the zebrafish GRCz10 release 86 genome assembly. Bowtie version1.1.1 was used with parameter \u2013e 200; UMI quantification was used with parameter \u2013u 2 counts were ignored from UMIs split between more than 2 genes. genomic DNA GESTALT and scGESTALT libraries were processed using a custom pipeline available at https://github.com/shendurelab/Cas9FateMapping Genome build: GRCz10 CSV files for transcriptome data were generated using the inDrops pipeline. Each column in the CSV files contains a cell identifier and each row contains expression values for genes. Txt files for genomic DNA GESTALT libraries *allReadCounts contain lineage barcode sequences HMID column for each cell and their proportion in the sequenced libraries. scGESTALT data *GestMaster.txt contains the inDrops cell identifiers CellBarcode and BarcodeKey that were used to match barcodes to transcriptomes. They also contain lineage barcode sequences HMID column for each cell with a corresponding inDrops single cell gene expression profile as well as the the t SNE cluster membership number ClusterIdent column Txt files ending in *stats.txt contain information about the each individually captured UMI or cell per sample. The barcode sequence aligned to a reference unedited sequence mergedRead column mutations at each target site target[X] columns and the edited sequences at each target site sequence[X] columns were used for downstream analysis. inDropsExpMatrix noQ txt file is the gene expression matrix for the full dataset. Columns are individual cells from different batches of whole brains f1 f2 f3 f4 f5 f6 or brain regions fore mid hind . fall.inDrops.Robj is the processed Seurat R object which can be loaded into R and explored.", "zebrafish brain", null, "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed and fragmented. The three prime fragments were reverse transcribed and prepared for sequencing Libraries were prepared as described in Zilionis et al. 2017 Nature Protocols PMID = 27929523", null, "tissue:brain|developmental stage:23 25dpf", "GSM2813975", "GSM2813975: DEW149 fore; Danio rerio; RNA Seq", "GSM2813975", null, "1", "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed and fragmented. The three prime fragments were reverse transcribed and prepared for sequencing Libraries were prepared as described in Zilionis et al. 2017 Nature Protocols PMID = 27929523", "GEO Accession:GSM2813975", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options: appendBCtoName", "DEW149_Lane2.sorted.fastq.gz", "fastq", 1324869432.0, 23093917.0, "GSM2813975 r2", "0:57.37", "A:367652083;C:249666726;G:278649220;T:428897643;N:3760", 57, null, null, null, 367652083, 249666726, 278649220, 428897643, 3760, "SRX3287405", "SRS2596879", "SRA619743", "GEO", "Harvard University", 1, 0.88017, null, 0.27604, null, 0.78658, null, 0.53022, null, 60, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43863, "SRR6176728", "SRX3287404", "SRS2596878", "SRP120009", "PRJNA414416", "Simultaneous single cell profiling of lineages and cell types in the vertebrate brain", "GSE105010", "Other", "The lineage relationships among the hundreds of cell types generated during development are difficult to reconstruct. A recent method GESTALT used CRISPR\u2013Cas9 barcode editing for large scale lineage tracing but was restricted to early development and did not identify cell types. Here we present scGESTALT which combines the lineage recording capabilities of GESTALT with cell type identification by single cell RNA sequencing. The method relies on an inducible system that enables barcodes to be edited at multiple time points capturing lineage information from later stages of development. Sequencing of 60 000 transcriptomes from the juvenile zebrafish brain identified >100 cell types and marker genes. Using these data we generate lineage trees with hundreds of branches that help uncover restrictions at the level of cell types brain regions and gene expression cascades during differentiation. scGESTALT can be applied to other multicellular organisms to simultaneously characterize molecular identities and lineage histories of thousands of cells during development and disease. Overall design: inDrops libraries of single cell transcriptomes and scGESTALT barcodes and genomic DNA GESTALT libraries", null, "pubmed:29608178", null, "DEW148 f6", "GSM2813974", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW148 f6", "Single cell RNA Sequencing data FASTQ files were processed using the inDrops.py bioinformatics pipeline available at https://github.com/indrops/indrops. Transcriptome libraries were mapped to a zebrafish reference built from a custom GTF file and the zebrafish GRCz10 release 86 genome assembly. Bowtie version1.1.1 was used with parameter \u2013e 200; UMI quantification was used with parameter \u2013u 2 counts were ignored from UMIs split between more than 2 genes. genomic DNA GESTALT and scGESTALT libraries were processed using a custom pipeline available at https://github.com/shendurelab/Cas9FateMapping Genome build: GRCz10 CSV files for transcriptome data were generated using the inDrops pipeline. Each column in the CSV files contains a cell identifier and each row contains expression values for genes. Txt files for genomic DNA GESTALT libraries *allReadCounts contain lineage barcode sequences HMID column for each cell and their proportion in the sequenced libraries. scGESTALT data *GestMaster.txt contains the inDrops cell identifiers CellBarcode and BarcodeKey that were used to match barcodes to transcriptomes. They also contain lineage barcode sequences HMID column for each cell with a corresponding inDrops single cell gene expression profile as well as the the t SNE cluster membership number ClusterIdent column Txt files ending in *stats.txt contain information about the each individually captured UMI or cell per sample. The barcode sequence aligned to a reference unedited sequence mergedRead column mutations at each target site target[X] columns and the edited sequences at each target site sequence[X] columns were used for downstream analysis. inDropsExpMatrix noQ txt file is the gene expression matrix for the full dataset. Columns are individual cells from different batches of whole brains f1 f2 f3 f4 f5 f6 or brain regions fore mid hind . fall.inDrops.Robj is the processed Seurat R object which can be loaded into R and explored.", "zebrafish brain", null, "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed and fragmented. The three prime fragments were reverse transcribed and prepared for sequencing Libraries were prepared as described in Zilionis et al. 2017 Nature Protocols PMID = 27929523", null, "tissue:brain|developmental stage:23 25dpf", "GSM2813974", "GSM2813974: DEW148 f6; Danio rerio; RNA Seq", "GSM2813974", null, "1", "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed and fragmented. The three prime fragments were reverse transcribed and prepared for sequencing Libraries were prepared as described in Zilionis et al. 2017 Nature Protocols PMID = 27929523", "GEO Accession:GSM2813974", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options: appendBCtoName", "DEW148_Lane1.sorted.fastq.gz", "fastq", 1783746165.0, 32647019.0, "GSM2813974 r1", "0:54.64", "A:492007833;C:343654151;G:370938043;T:577142407;N:3731", 54, null, null, null, 492007833, 343654151, 370938043, 577142407, 3731, "SRX3287404", "SRS2596878", "SRA619743", "GEO", "Harvard University", 1, 0.86946, null, 0.22807, null, 0.79462, null, 0.51961, null, 61, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43864, "SRR6176729", "SRX3287404", "SRS2596878", "SRP120009", "PRJNA414416", "Simultaneous single cell profiling of lineages and cell types in the vertebrate brain", "GSE105010", "Other", "The lineage relationships among the hundreds of cell types generated during development are difficult to reconstruct. A recent method GESTALT used CRISPR\u2013Cas9 barcode editing for large scale lineage tracing but was restricted to early development and did not identify cell types. Here we present scGESTALT which combines the lineage recording capabilities of GESTALT with cell type identification by single cell RNA sequencing. The method relies on an inducible system that enables barcodes to be edited at multiple time points capturing lineage information from later stages of development. Sequencing of 60 000 transcriptomes from the juvenile zebrafish brain identified >100 cell types and marker genes. Using these data we generate lineage trees with hundreds of branches that help uncover restrictions at the level of cell types brain regions and gene expression cascades during differentiation. scGESTALT can be applied to other multicellular organisms to simultaneously characterize molecular identities and lineage histories of thousands of cells during development and disease. Overall design: inDrops libraries of single cell transcriptomes and scGESTALT barcodes and genomic DNA GESTALT libraries", null, "pubmed:29608178", null, "DEW148 f6", "GSM2813974", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW148 f6", "Single cell RNA Sequencing data FASTQ files were processed using the inDrops.py bioinformatics pipeline available at https://github.com/indrops/indrops. Transcriptome libraries were mapped to a zebrafish reference built from a custom GTF file and the zebrafish GRCz10 release 86 genome assembly. Bowtie version1.1.1 was used with parameter \u2013e 200; UMI quantification was used with parameter \u2013u 2 counts were ignored from UMIs split between more than 2 genes. genomic DNA GESTALT and scGESTALT libraries were processed using a custom pipeline available at https://github.com/shendurelab/Cas9FateMapping Genome build: GRCz10 CSV files for transcriptome data were generated using the inDrops pipeline. Each column in the CSV files contains a cell identifier and each row contains expression values for genes. Txt files for genomic DNA GESTALT libraries *allReadCounts contain lineage barcode sequences HMID column for each cell and their proportion in the sequenced libraries. scGESTALT data *GestMaster.txt contains the inDrops cell identifiers CellBarcode and BarcodeKey that were used to match barcodes to transcriptomes. They also contain lineage barcode sequences HMID column for each cell with a corresponding inDrops single cell gene expression profile as well as the the t SNE cluster membership number ClusterIdent column Txt files ending in *stats.txt contain information about the each individually captured UMI or cell per sample. The barcode sequence aligned to a reference unedited sequence mergedRead column mutations at each target site target[X] columns and the edited sequences at each target site sequence[X] columns were used for downstream analysis. inDropsExpMatrix noQ txt file is the gene expression matrix for the full dataset. Columns are individual cells from different batches of whole brains f1 f2 f3 f4 f5 f6 or brain regions fore mid hind . fall.inDrops.Robj is the processed Seurat R object which can be loaded into R and explored.", "zebrafish brain", null, "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed and fragmented. The three prime fragments were reverse transcribed and prepared for sequencing Libraries were prepared as described in Zilionis et al. 2017 Nature Protocols PMID = 27929523", null, "tissue:brain|developmental stage:23 25dpf", "GSM2813974", "GSM2813974: DEW148 f6; Danio rerio; RNA Seq", "GSM2813974", null, "1", "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed and fragmented. The three prime fragments were reverse transcribed and prepared for sequencing Libraries were prepared as described in Zilionis et al. 2017 Nature Protocols PMID = 27929523", "GEO Accession:GSM2813974", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options: appendBCtoName", "DEW148_Lane2.sorted.fastq.gz", "fastq", 2321976093.0, 39701872.0, "GSM2813974 r2", "0:58.49", "A:644394074;C:446370620;G:489186161;T:742017510;N:7728", 58, null, null, null, 644394074, 446370620, 489186161, 742017510, 7728, "SRX3287404", "SRS2596878", "SRA619743", "GEO", "Harvard University", 1, 0.8902, null, 0.22908, null, 0.78689, null, 0.52036, null, 61, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43865, "SRR6176726", "SRX3287403", "SRS2596877", "SRP120009", "PRJNA414416", "Simultaneous single cell profiling of lineages and cell types in the vertebrate brain", "GSE105010", "Other", "The lineage relationships among the hundreds of cell types generated during development are difficult to reconstruct. A recent method GESTALT used CRISPR\u2013Cas9 barcode editing for large scale lineage tracing but was restricted to early development and did not identify cell types. Here we present scGESTALT which combines the lineage recording capabilities of GESTALT with cell type identification by single cell RNA sequencing. The method relies on an inducible system that enables barcodes to be edited at multiple time points capturing lineage information from later stages of development. Sequencing of 60 000 transcriptomes from the juvenile zebrafish brain identified >100 cell types and marker genes. Using these data we generate lineage trees with hundreds of branches that help uncover restrictions at the level of cell types brain regions and gene expression cascades during differentiation. scGESTALT can be applied to other multicellular organisms to simultaneously characterize molecular identities and lineage histories of thousands of cells during development and disease. Overall design: inDrops libraries of single cell transcriptomes and scGESTALT barcodes and genomic DNA GESTALT libraries", null, "pubmed:29608178", null, "DEW147 f6", "GSM2813973", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW147 f6", "Single cell RNA Sequencing data FASTQ files were processed using the inDrops.py bioinformatics pipeline available at https://github.com/indrops/indrops. Transcriptome libraries were mapped to a zebrafish reference built from a custom GTF file and the zebrafish GRCz10 release 86 genome assembly. Bowtie version1.1.1 was used with parameter \u2013e 200; UMI quantification was used with parameter \u2013u 2 counts were ignored from UMIs split between more than 2 genes. genomic DNA GESTALT and scGESTALT libraries were processed using a custom pipeline available at https://github.com/shendurelab/Cas9FateMapping Genome build: GRCz10 CSV files for transcriptome data were generated using the inDrops pipeline. Each column in the CSV files contains a cell identifier and each row contains expression values for genes. Txt files for genomic DNA GESTALT libraries *allReadCounts contain lineage barcode sequences HMID column for each cell and their proportion in the sequenced libraries. scGESTALT data *GestMaster.txt contains the inDrops cell identifiers CellBarcode and BarcodeKey that were used to match barcodes to transcriptomes. They also contain lineage barcode sequences HMID column for each cell with a corresponding inDrops single cell gene expression profile as well as the the t SNE cluster membership number ClusterIdent column Txt files ending in *stats.txt contain information about the each individually captured UMI or cell per sample. The barcode sequence aligned to a reference unedited sequence mergedRead column mutations at each target site target[X] columns and the edited sequences at each target site sequence[X] columns were used for downstream analysis. inDropsExpMatrix noQ txt file is the gene expression matrix for the full dataset. Columns are individual cells from different batches of whole brains f1 f2 f3 f4 f5 f6 or brain regions fore mid hind . fall.inDrops.Robj is the processed Seurat R object which can be loaded into R and explored.", "zebrafish brain", null, "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed and fragmented. The three prime fragments were reverse transcribed and prepared for sequencing Libraries were prepared as described in Zilionis et al. 2017 Nature Protocols PMID = 27929523", null, "tissue:brain|developmental stage:23 25dpf", "GSM2813973", "GSM2813973: DEW147 f6; Danio rerio; RNA Seq", "GSM2813973", null, "1", "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed and fragmented. The three prime fragments were reverse transcribed and prepared for sequencing Libraries were prepared as described in Zilionis et al. 2017 Nature Protocols PMID = 27929523", "GEO Accession:GSM2813973", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options: appendBCtoName", "DEW147_Lane1.sorted.fastq.gz", "fastq", 1861151726.0, 33960188.0, "GSM2813973 r1", "0:54.80", "A:514475433;C:356385455;G:385168377;T:605118287;N:4174", 54, null, null, null, 514475433, 356385455, 385168377, 605118287, 4174, "SRX3287403", "SRS2596877", "SRA619743", "GEO", "Harvard University", 1, 0.86477, null, 0.23182, null, 0.79454, null, 0.51967, null, 41, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43866, "SRR6176727", "SRX3287403", "SRS2596877", "SRP120009", "PRJNA414416", "Simultaneous single cell profiling of lineages and cell types in the vertebrate brain", "GSE105010", "Other", "The lineage relationships among the hundreds of cell types generated during development are difficult to reconstruct. A recent method GESTALT used CRISPR\u2013Cas9 barcode editing for large scale lineage tracing but was restricted to early development and did not identify cell types. Here we present scGESTALT which combines the lineage recording capabilities of GESTALT with cell type identification by single cell RNA sequencing. The method relies on an inducible system that enables barcodes to be edited at multiple time points capturing lineage information from later stages of development. Sequencing of 60 000 transcriptomes from the juvenile zebrafish brain identified >100 cell types and marker genes. Using these data we generate lineage trees with hundreds of branches that help uncover restrictions at the level of cell types brain regions and gene expression cascades during differentiation. scGESTALT can be applied to other multicellular organisms to simultaneously characterize molecular identities and lineage histories of thousands of cells during development and disease. Overall design: inDrops libraries of single cell transcriptomes and scGESTALT barcodes and genomic DNA GESTALT libraries", null, "pubmed:29608178", null, "DEW147 f6", "GSM2813973", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW147 f6", "Single cell RNA Sequencing data FASTQ files were processed using the inDrops.py bioinformatics pipeline available at https://github.com/indrops/indrops. Transcriptome libraries were mapped to a zebrafish reference built from a custom GTF file and the zebrafish GRCz10 release 86 genome assembly. Bowtie version1.1.1 was used with parameter \u2013e 200; UMI quantification was used with parameter \u2013u 2 counts were ignored from UMIs split between more than 2 genes. genomic DNA GESTALT and scGESTALT libraries were processed using a custom pipeline available at https://github.com/shendurelab/Cas9FateMapping Genome build: GRCz10 CSV files for transcriptome data were generated using the inDrops pipeline. Each column in the CSV files contains a cell identifier and each row contains expression values for genes. Txt files for genomic DNA GESTALT libraries *allReadCounts contain lineage barcode sequences HMID column for each cell and their proportion in the sequenced libraries. scGESTALT data *GestMaster.txt contains the inDrops cell identifiers CellBarcode and BarcodeKey that were used to match barcodes to transcriptomes. They also contain lineage barcode sequences HMID column for each cell with a corresponding inDrops single cell gene expression profile as well as the the t SNE cluster membership number ClusterIdent column Txt files ending in *stats.txt contain information about the each individually captured UMI or cell per sample. The barcode sequence aligned to a reference unedited sequence mergedRead column mutations at each target site target[X] columns and the edited sequences at each target site sequence[X] columns were used for downstream analysis. inDropsExpMatrix noQ txt file is the gene expression matrix for the full dataset. Columns are individual cells from different batches of whole brains f1 f2 f3 f4 f5 f6 or brain regions fore mid hind . fall.inDrops.Robj is the processed Seurat R object which can be loaded into R and explored.", "zebrafish brain", null, "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed and fragmented. The three prime fragments were reverse transcribed and prepared for sequencing Libraries were prepared as described in Zilionis et al. 2017 Nature Protocols PMID = 27929523", null, "tissue:brain|developmental stage:23 25dpf", "GSM2813973", "GSM2813973: DEW147 f6; Danio rerio; RNA Seq", "GSM2813973", null, "1", "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed and fragmented. The three prime fragments were reverse transcribed and prepared for sequencing Libraries were prepared as described in Zilionis et al. 2017 Nature Protocols PMID = 27929523", "GEO Accession:GSM2813973", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options: appendBCtoName", "DEW147_Lane2.sorted.fastq.gz", "fastq", 2384082430.0, 40711661.0, "GSM2813973 r2", "0:58.56", "A:662878220;C:455374350;G:499509285;T:766312566;N:8009", 58, null, null, null, 662878220, 455374350, 499509285, 766312566, 8009, "SRX3287403", "SRS2596877", "SRA619743", "GEO", "Harvard University", 1, 0.88512, null, 0.23293, null, 0.7878, null, 0.51475, null, 61, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43867, "SRR6176724", "SRX3287402", "SRS2596876", "SRP120009", "PRJNA414416", "Simultaneous single cell profiling of lineages and cell types in the vertebrate brain", "GSE105010", "Other", "The lineage relationships among the hundreds of cell types generated during development are difficult to reconstruct. A recent method GESTALT used CRISPR\u2013Cas9 barcode editing for large scale lineage tracing but was restricted to early development and did not identify cell types. Here we present scGESTALT which combines the lineage recording capabilities of GESTALT with cell type identification by single cell RNA sequencing. The method relies on an inducible system that enables barcodes to be edited at multiple time points capturing lineage information from later stages of development. Sequencing of 60 000 transcriptomes from the juvenile zebrafish brain identified >100 cell types and marker genes. Using these data we generate lineage trees with hundreds of branches that help uncover restrictions at the level of cell types brain regions and gene expression cascades during differentiation. scGESTALT can be applied to other multicellular organisms to simultaneously characterize molecular identities and lineage histories of thousands of cells during development and disease. Overall design: inDrops libraries of single cell transcriptomes and scGESTALT barcodes and genomic DNA GESTALT libraries", null, "pubmed:29608178", null, "DEW146 f6", "GSM2813972", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW146 f6", "Single cell RNA Sequencing data FASTQ files were processed using the inDrops.py bioinformatics pipeline available at https://github.com/indrops/indrops. Transcriptome libraries were mapped to a zebrafish reference built from a custom GTF file and the zebrafish GRCz10 release 86 genome assembly. Bowtie version1.1.1 was used with parameter \u2013e 200; UMI quantification was used with parameter \u2013u 2 counts were ignored from UMIs split between more than 2 genes. genomic DNA GESTALT and scGESTALT libraries were processed using a custom pipeline available at https://github.com/shendurelab/Cas9FateMapping Genome build: GRCz10 CSV files for transcriptome data were generated using the inDrops pipeline. Each column in the CSV files contains a cell identifier and each row contains expression values for genes. Txt files for genomic DNA GESTALT libraries *allReadCounts contain lineage barcode sequences HMID column for each cell and their proportion in the sequenced libraries. scGESTALT data *GestMaster.txt contains the inDrops cell identifiers CellBarcode and BarcodeKey that were used to match barcodes to transcriptomes. They also contain lineage barcode sequences HMID column for each cell with a corresponding inDrops single cell gene expression profile as well as the the t SNE cluster membership number ClusterIdent column Txt files ending in *stats.txt contain information about the each individually captured UMI or cell per sample. The barcode sequence aligned to a reference unedited sequence mergedRead column mutations at each target site target[X] columns and the edited sequences at each target site sequence[X] columns were used for downstream analysis. inDropsExpMatrix noQ txt file is the gene expression matrix for the full dataset. Columns are individual cells from different batches of whole brains f1 f2 f3 f4 f5 f6 or brain regions fore mid hind . fall.inDrops.Robj is the processed Seurat R object which can be loaded into R and explored.", "zebrafish brain", null, "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed and fragmented. The three prime fragments were reverse transcribed and prepared for sequencing Libraries were prepared as described in Zilionis et al. 2017 Nature Protocols PMID = 27929523", null, "tissue:brain|developmental stage:23 25dpf", "GSM2813972", "GSM2813972: DEW146 f6; Danio rerio; RNA Seq", "GSM2813972", null, "1", "Single cell suspensions were processed through inDrops to generate single cell cDNA libraries. cDNAs were in vitro transcribed and fragmented. The three prime fragments were reverse transcribed and prepared for sequencing Libraries were prepared as described in Zilionis et al. 2017 Nature Protocols PMID = 27929523", "GEO Accession:GSM2813972", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options: appendBCtoName", "DEW146_Lane1.sorted.fastq.gz", "fastq", 1879106765.0, 34463595.0, "GSM2813972 r1", "0:54.52", "A:518564123;C:360410554;G:392630549;T:607497754;N:3785", 54, null, null, null, 518564123, 360410554, 392630549, 607497754, 3785, "SRX3287402", "SRS2596876", "SRA619743", "GEO", "Harvard University", 1, 0.868, null, 0.22669, null, 0.79506, null, 0.50915, null, 55, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"]], "truncated": false, "filtered_table_rows_count": 369, "expanded_columns": [], "expandable_columns": [], "columns": ["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"], "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 \"experiment.library_source\" = :p0 and \"technology\" = :p1 order by rowid limit 101", "params": {"p0": "TRANSCRIPTOMIC", "p1": "indrops"}}, "facet_results": {"experiment.library_strategy": {"name": "experiment.library_strategy", "type": "column", "hideable": false, "toggle_url": "/metadata/run_metadata.json?experiment.library_source=TRANSCRIPTOMIC&technology=indrops", "results": [{"value": "RNA-Seq", "label": "RNA-Seq", "count": 308, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?experiment.library_source=TRANSCRIPTOMIC&technology=indrops&experiment.library_strategy=RNA-Seq", "selected": false}, {"value": "OTHER", "label": "OTHER", "count": 61, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?experiment.library_source=TRANSCRIPTOMIC&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?experiment.library_source=TRANSCRIPTOMIC&technology=indrops", "results": [{"value": "TRANSCRIPTOMIC", "label": "TRANSCRIPTOMIC", "count": 369, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?technology=indrops", "selected": true}], "truncated": false}, "experiment.library_selection": {"name": "experiment.library_selection", "type": "column", "hideable": false, "toggle_url": "/metadata/run_metadata.json?experiment.library_source=TRANSCRIPTOMIC&technology=indrops", "results": [{"value": "cDNA", "label": "cDNA", "count": 308, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?experiment.library_source=TRANSCRIPTOMIC&technology=indrops&experiment.library_selection=cDNA", "selected": false}, {"value": "other", "label": "other", "count": 61, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?experiment.library_source=TRANSCRIPTOMIC&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?experiment.library_source=TRANSCRIPTOMIC&technology=indrops", "results": [{"value": "SINGLE", "label": "SINGLE", "count": 253, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?experiment.library_source=TRANSCRIPTOMIC&technology=indrops&experiment.library_layout=SINGLE", "selected": false}, {"value": "PAIRED", "label": "PAIRED", "count": 116, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?experiment.library_source=TRANSCRIPTOMIC&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?experiment.library_source=TRANSCRIPTOMIC&technology=indrops", "results": [{"value": "ILLUMINA", "label": "ILLUMINA", "count": 369, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?experiment.library_source=TRANSCRIPTOMIC&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?experiment.library_source=TRANSCRIPTOMIC&technology=indrops", "results": [{"value": "Embryo", "label": "Embryo", "count": 152, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?experiment.library_source=TRANSCRIPTOMIC&technology=indrops&devstage_curation_coarse=Embryo", "selected": false}, {"value": "Larval", "label": "Larval", "count": 145, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?experiment.library_source=TRANSCRIPTOMIC&technology=indrops&devstage_curation_coarse=Larval", "selected": false}, {"value": "Juvenile", "label": "Juvenile", "count": 33, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?experiment.library_source=TRANSCRIPTOMIC&technology=indrops&devstage_curation_coarse=Juvenile", "selected": false}, {"value": "Adult", "label": "Adult", "count": 25, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?experiment.library_source=TRANSCRIPTOMIC&technology=indrops&devstage_curation_coarse=Adult", "selected": false}, {"value": "Undetermined", "label": "Undetermined", "count": 14, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?experiment.library_source=TRANSCRIPTOMIC&technology=indrops&devstage_curation_coarse=Undetermined", "selected": false}], "truncated": false}, "devstage_curation": {"name": "devstage_curation", "type": "column", "hideable": false, "toggle_url": "/metadata/run_metadata.json?experiment.library_source=TRANSCRIPTOMIC&technology=indrops", "results": [{"value": "Larval", "label": "Larval", "count": 145, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?experiment.library_source=TRANSCRIPTOMIC&technology=indrops&devstage_curation=Larval", "selected": false}, {"value": "Multi-stage", "label": "Multi-stage", "count": 58, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?experiment.library_source=TRANSCRIPTOMIC&technology=indrops&devstage_curation=Multi-stage", "selected": false}, {"value": "Pharyngula", "label": "Pharyngula", "count": 51, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?experiment.library_source=TRANSCRIPTOMIC&technology=indrops&devstage_curation=Pharyngula", "selected": false}, {"value": "Juvenile", "label": "Juvenile", "count": 33, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?experiment.library_source=TRANSCRIPTOMIC&technology=indrops&devstage_curation=Juvenile", "selected": false}, {"value": "Undetermined", "label": "Undetermined", "count": 30, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?experiment.library_source=TRANSCRIPTOMIC&technology=indrops&devstage_curation=Undetermined", "selected": false}, {"value": "Segmentation", "label": "Segmentation", "count": 29, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?experiment.library_source=TRANSCRIPTOMIC&technology=indrops&devstage_curation=Segmentation", "selected": false}, {"value": "Gastrula", "label": "Gastrula", "count": 12, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?experiment.library_source=TRANSCRIPTOMIC&technology=indrops&devstage_curation=Gastrula", "selected": false}, {"value": "Adult", "label": "Adult", "count": 9, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?experiment.library_source=TRANSCRIPTOMIC&technology=indrops&devstage_curation=Adult", "selected": false}, {"value": "Blastula", "label": "Blastula", "count": 2, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?experiment.library_source=TRANSCRIPTOMIC&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?experiment.library_source=TRANSCRIPTOMIC&technology=indrops", "results": [{"value": "All anatomical structures", "label": "All anatomical structures", "count": 154, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?experiment.library_source=TRANSCRIPTOMIC&technology=indrops&tissue_curation_coarse=All+anatomical+structures", "selected": false}, {"value": "Nervous System", "label": "Nervous System", "count": 141, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?experiment.library_source=TRANSCRIPTOMIC&technology=indrops&tissue_curation_coarse=Nervous+System", "selected": false}, {"value": "Multi-system", "label": "Multi-system", "count": 35, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?experiment.library_source=TRANSCRIPTOMIC&technology=indrops&tissue_curation_coarse=Multi-system", "selected": false}, {"value": "Renal System", "label": "Renal System", "count": 25, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?experiment.library_source=TRANSCRIPTOMIC&technology=indrops&tissue_curation_coarse=Renal+System", "selected": false}, {"value": "Cancer or Tumor", "label": "Cancer or Tumor", "count": 14, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?experiment.library_source=TRANSCRIPTOMIC&technology=indrops&tissue_curation_coarse=Cancer+or+Tumor", "selected": false}], "truncated": false}, "tissue_curation": {"name": "tissue_curation", "type": "column", "hideable": false, "toggle_url": "/metadata/run_metadata.json?experiment.library_source=TRANSCRIPTOMIC&technology=indrops", "results": [{"value": "Brain", "label": "Brain", "count": 141, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?experiment.library_source=TRANSCRIPTOMIC&technology=indrops&tissue_curation=Brain", "selected": false}, {"value": "Embryo Imprecise", "label": "Embryo Imprecise", "count": 94, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?experiment.library_source=TRANSCRIPTOMIC&technology=indrops&tissue_curation=Embryo+Imprecise", "selected": false}, {"value": "Whole Organism", "label": "Whole Organism", "count": 60, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?experiment.library_source=TRANSCRIPTOMIC&technology=indrops&tissue_curation=Whole+Organism", "selected": false}, {"value": "Multi-tissue", "label": "Multi-tissue", "count": 33, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?experiment.library_source=TRANSCRIPTOMIC&technology=indrops&tissue_curation=Multi-tissue", "selected": false}, {"value": "Kidney", "label": "Kidney", "count": 16, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?experiment.library_source=TRANSCRIPTOMIC&technology=indrops&tissue_curation=Kidney", "selected": false}, {"value": "Cancer or Tumor", "label": "Cancer or Tumor", "count": 14, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?experiment.library_source=TRANSCRIPTOMIC&technology=indrops&tissue_curation=Cancer+or+Tumor", "selected": false}, {"value": "Marrow", "label": "Marrow", "count": 9, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?experiment.library_source=TRANSCRIPTOMIC&technology=indrops&tissue_curation=Marrow", "selected": false}, {"value": "Tail", "label": "Tail", "count": 2, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?experiment.library_source=TRANSCRIPTOMIC&technology=indrops&tissue_curation=Tail", "selected": false}], "truncated": false}, "technology": {"name": "technology", "type": "column", "hideable": false, "toggle_url": "/metadata/run_metadata.json?experiment.library_source=TRANSCRIPTOMIC&technology=indrops", "results": [{"value": "indrops", "label": "indrops", "count": 369, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?experiment.library_source=TRANSCRIPTOMIC", "selected": true}], "truncated": false}}, "suggested_facets": [], "next": "43867", "next_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?experiment.library_source=TRANSCRIPTOMIC&technology=indrops&_next=43867", "private": false, "allow_execute_sql": true, "query_ms": 127.11964499612805}