{"database": "metadata", "table": "run_metadata", "is_view": false, "human_description_en": "where experiment.library_layout = \"PAIRED\", 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"], [43939, "SRR6176649", "SRX3287366", "SRS2596840", "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, "DEW067 f2", "GSM2813936", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW067 f2", "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", "GSM2813936", "GSM2813936: DEW067 f2; Danio rerio; RNA Seq", "GSM2813936", 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:GSM2813936", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options:  appendBCtoName", "FC1_DEW-067_S10.R1.fastq.gz FC1_DEW-067_S10.R2.fastq.gz", "fastq fastq", 2551096506.0, 30568258.0, "GSM2813936 r1", null, "A:557861215;C:486414729;G:614056373;T:892106619;N:657570", null, null, null, null, 557861215, 486414729, 614056373, 892106619, 657570, "SRX3287366", "SRS2596840", "SRA619743", "GEO", "Harvard University", 2, 0.74967, 0.00628, 0.22106, 0.006, 0.79527, 0.99945, 0.55023, 0.46875, 34, 49, "B", "T", "mate2 technical by mapping diff", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43940, "SRR6176650", "SRX3287366", "SRS2596840", "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, "DEW067 f2", "GSM2813936", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW067 f2", "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", "GSM2813936", "GSM2813936: DEW067 f2; Danio rerio; RNA Seq", "GSM2813936", 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:GSM2813936", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options:  appendBCtoName", "FC2_DEW-067_S10.R1.fastq.gz FC2_DEW-067_S10.R2.fastq.gz", "fastq fastq", 1155281120.0, 13849592.0, "GSM2813936 r2", null, "A:233187378;C:202743748;G:364218355;T:354503618;N:628021", null, null, null, null, 233187378, 202743748, 364218355, 354503618, 628021, "SRX3287366", "SRS2596840", "SRA619743", "GEO", "Harvard University", 2, 0.75419, 0.02424, 0.22148, 0.02238, 0.79415, 0.99943, 0.55145, 0.9, 34, 50, "B", "T", "mate2 technical by mapping diff", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43941, "SRR6176651", "SRX3287366", "SRS2596840", "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, "DEW067 f2", "GSM2813936", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW067 f2", "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", "GSM2813936", "GSM2813936: DEW067 f2; Danio rerio; RNA Seq", "GSM2813936", 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:GSM2813936", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options:  appendBCtoName", "FC3_DEW-067_S10.R1.fastq.gz FC3_DEW-067_S10.R2.fastq.gz", "fastq fastq", 2630905456.0, 31531644.0, "GSM2813936 r3", null, "A:571354506;C:501012015;G:621212959;T:936052979;N:1272997", null, null, null, null, 571354506, 501012015, 621212959, 936052979, 1272997, "SRX3287366", "SRS2596840", "SRA619743", "GEO", "Harvard University", 2, 0.75366, 0.0038, 0.22017, 0.00351, 0.79265, 0.99931, 0.5547, 0.63414, 33, 50, "B", "T", "mate2 technical by mapping diff", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43942, "SRR6176652", "SRX3287366", "SRS2596840", "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, "DEW067 f2", "GSM2813936", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW067 f2", "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", "GSM2813936", "GSM2813936: DEW067 f2; Danio rerio; RNA Seq", "GSM2813936", 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:GSM2813936", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options:  appendBCtoName", "FC4_DEW-067_S10.R1.fastq.gz FC4_DEW-067_S10.R2.fastq.gz", "fastq fastq", 2426720147.0, 29074926.0, "GSM2813936 r4", null, "A:521871975;C:458708124;G:576717935;T:869109171;N:312942", null, null, null, null, 521871975, 458708124, 576717935, 869109171, 312942, "SRX3287366", "SRS2596840", "SRA619743", "GEO", "Harvard University", 2, 0.75679, 0.00616, 0.22081, 0.00577, 0.79074, 0.99896, 0.55926, 0.67692, 34, 50, "B", "T", "mate2 technical by mapping diff", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43943, "SRR6176653", "SRX3287366", "SRS2596840", "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, "DEW067 f2", "GSM2813936", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW067 f2", "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", "GSM2813936", "GSM2813936: DEW067 f2; Danio rerio; RNA Seq", "GSM2813936", 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:GSM2813936", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options:  appendBCtoName", "FC5_DEW-067_S4.R1.fastq.gz FC5_DEW-067_S4.R2.fastq.gz", "fastq fastq", 7689824768.0, 92165385.0, "GSM2813936 r5", null, "A:1694218914;C:1478360575;G:1791783776;T:2724399126;N:1062377", null, null, null, null, 1694218914, 1478360575, 1791783776, 2724399126, 1062377, "SRX3287366", "SRS2596840", "SRA619743", "GEO", "Harvard University", 2, 0.75345, 0.00154, 0.22254, 0.00137, 0.79263, 0.99967, 0.55441, 0.57142, 33, 50, "B", "T", "mate2 technical by mapping diff", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43944, "SRR6176644", "SRX3287365", "SRS2596839", "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, "DEW066 f2", "GSM2813935", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW066 f2", "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", "GSM2813935", "GSM2813935: DEW066 f2; Danio rerio; RNA Seq", "GSM2813935", 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:GSM2813935", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options:  appendBCtoName", "FC1_DEW-066_S9.R1.fastq.gz FC1_DEW-066_S9.R2.fastq.gz", "fastq fastq", 2140044898.0, 25651167.0, "GSM2813935 r1", null, "A:467615827;C:408349284;G:516714028;T:746812741;N:553018", null, null, null, null, 467615827, 408349284, 516714028, 746812741, 553018, "SRX3287365", "SRS2596839", "SRA619743", "GEO", "Harvard University", 2, 0.74758, 0.00524, 0.2137, 0.00493, 0.7987, 0.99941, 0.55876, 0.52631, 34, 49, "B", "T", "mate2 technical by mapping diff", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43945, "SRR6176645", "SRX3287365", "SRS2596839", "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, "DEW066 f2", "GSM2813935", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW066 f2", "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", "GSM2813935", "GSM2813935: DEW066 f2; Danio rerio; RNA Seq", "GSM2813935", 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:GSM2813935", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options:  appendBCtoName", "FC2_DEW-066_S9.R1.fastq.gz FC2_DEW-066_S9.R2.fastq.gz", "fastq fastq", 965058666.0, 11574298.0, "GSM2813935 r2", null, "A:195003483;C:170100194;G:303694091;T:295746532;N:514366", null, null, null, null, 195003483, 170100194, 303694091, 295746532, 514366, "SRX3287365", "SRS2596839", "SRA619743", "GEO", "Harvard University", 2, 0.75025, 0.01899, 0.21414, 0.01762, 0.7977, 0.99943, 0.5594, 0.24832, 34, 50, "B", "T", "mate2 technical by mapping diff", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43946, "SRR6176646", "SRX3287365", "SRS2596839", "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, "DEW066 f2", "GSM2813935", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW066 f2", "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", "GSM2813935", "GSM2813935: DEW066 f2; Danio rerio; RNA Seq", "GSM2813935", 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:GSM2813935", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options:  appendBCtoName", "FC3_DEW-066_S9.R1.fastq.gz FC3_DEW-066_S9.R2.fastq.gz", "fastq fastq", 2086254612.0, 25012693.0, "GSM2813935 r3", null, "A:452988540;C:397479617;G:494445479;T:740347977;N:992999", null, null, null, null, 452988540, 397479617, 494445479, 740347977, 992999, "SRX3287365", "SRS2596839", "SRA619743", "GEO", "Harvard University", 2, 0.75183, 0.00319, 0.21451, 0.0029, 0.79429, 0.99935, 0.56364, 0.58536, 34, 50, "B", "T", "mate2 technical by mapping diff", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43947, "SRR6176647", "SRX3287365", "SRS2596839", "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, "DEW066 f2", "GSM2813935", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW066 f2", "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", "GSM2813935", "GSM2813935: DEW066 f2; Danio rerio; RNA Seq", "GSM2813935", 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:GSM2813935", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options:  appendBCtoName", "FC4_DEW-066_S9.R1.fastq.gz FC4_DEW-066_S9.R2.fastq.gz", "fastq fastq", 1923868374.0, 23057073.0, "GSM2813935 r4", null, "A:413966332;C:363922629;G:458666692;T:687070548;N:242173", null, null, null, null, 413966332, 363922629, 458666692, 687070548, 242173, "SRX3287365", "SRS2596839", "SRA619743", "GEO", "Harvard University", 2, 0.75485, 0.00489, 0.21313, 0.00454, 0.79281, 0.99908, 0.55638, 0.54237, 33, 50, "B", "T", "mate2 technical by mapping diff", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43948, "SRR6176648", "SRX3287365", "SRS2596839", "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, "DEW066 f2", "GSM2813935", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW066 f2", "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", "GSM2813935", "GSM2813935: DEW066 f2; Danio rerio; RNA Seq", "GSM2813935", 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:GSM2813935", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options:  appendBCtoName", "FC5_DEW-066_S3.R1.fastq.gz FC5_DEW-066_S3.R2.fastq.gz", "fastq fastq", 6104866273.0, 73196289.0, "GSM2813935 r5", null, "A:1345028270;C:1173765932;G:1427797225;T:2157425704;N:849142", null, null, null, null, 1345028270, 1173765932, 1427797225, 2157425704, 849142, "SRX3287365", "SRS2596839", "SRA619743", "GEO", "Harvard University", 2, 0.75337, 0.00127, 0.21518, 0.00111, 0.79437, 0.99961, 0.54968, 0.65, 33, 49, "B", "T", "mate2 technical by mapping diff", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43949, "SRR6176639", "SRX3287364", "SRS2596838", "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, "DEW065 f2", "GSM2813934", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW065 f2", "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", "GSM2813934", "GSM2813934: DEW065 f2; Danio rerio; RNA Seq", "GSM2813934", 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:GSM2813934", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options:  appendBCtoName", "FC1_DEW-065_S8.R1.fastq.gz FC1_DEW-065_S8.R2.fastq.gz", "fastq fastq", 2299056187.0, 27557923.0, "GSM2813934 r1", null, "A:501686139;C:441327292;G:550895560;T:804557888;N:589308", null, null, null, null, 501686139, 441327292, 550895560, 804557888, 589308, "SRX3287364", "SRS2596838", "SRA619743", "GEO", "Harvard University", 2, 0.73689, 0.00588, 0.20924, 0.00551, 0.80273, 0.99926, 0.5692, 0.55555, 33, 49, "B", "T", "mate2 technical by mapping diff", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43950, "SRR6176640", "SRX3287364", "SRS2596838", "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, "DEW065 f2", "GSM2813934", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW065 f2", "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", "GSM2813934", "GSM2813934: DEW065 f2; Danio rerio; RNA Seq", "GSM2813934", 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:GSM2813934", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options:  appendBCtoName", "FC2_DEW-065_S8.R1.fastq.gz FC2_DEW-065_S8.R2.fastq.gz", "fastq fastq", 1037253750.0, 12441178.0, "GSM2813934 r2", null, "A:209154151;C:183891879;G:324728467;T:318916935;N:562318", null, null, null, null, 209154151, 183891879, 324728467, 318916935, 562318, "SRX3287364", "SRS2596838", "SRA619743", "GEO", "Harvard University", 2, 0.74123, 0.01962, 0.21258, 0.01799, 0.80318, 0.99926, 0.5667, 0.8983, 34, 50, "B", "T", "mate2 technical by mapping diff", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43951, "SRR6176641", "SRX3287364", "SRS2596838", "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, "DEW065 f2", "GSM2813934", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW065 f2", "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", "GSM2813934", "GSM2813934: DEW065 f2; Danio rerio; RNA Seq", "GSM2813934", 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:GSM2813934", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options:  appendBCtoName", "FC3_DEW-065_S8.R1.fastq.gz FC3_DEW-065_S8.R2.fastq.gz", "fastq fastq", 2440058543.0, 29255489.0, "GSM2813934 r3", null, "A:529090031;C:467802013;G:573700898;T:868289954;N:1175647", null, null, null, null, 529090031, 467802013, 573700898, 868289954, 1175647, "SRX3287364", "SRS2596838", "SRA619743", "GEO", "Harvard University", 2, 0.74341, 0.00364, 0.21329, 0.00338, 0.79983, 0.99949, 0.56272, 0.45945, 33, 50, "B", "T", "mate2 technical by mapping diff", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43952, "SRR6176642", "SRX3287364", "SRS2596838", "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, "DEW065 f2", "GSM2813934", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW065 f2", "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", "GSM2813934", "GSM2813934: DEW065 f2; Danio rerio; RNA Seq", "GSM2813934", 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:GSM2813934", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options:  appendBCtoName", "FC4_DEW-065_S8.R1.fastq.gz FC4_DEW-065_S8.R2.fastq.gz", "fastq fastq", 2248915479.0, 26953029.0, "GSM2813934 r4", null, "A:482877461;C:428438228;G:531631238;T:805679337;N:289215", null, null, null, null, 482877461, 428438228, 531631238, 805679337, 289215, "SRX3287364", "SRS2596838", "SRA619743", "GEO", "Harvard University", 2, 0.74561, 0.00511, 0.21378, 0.00469, 0.79876, 0.99898, 0.56089, 0.54285, 33, 50, "B", "T", "mate2 technical by mapping diff", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43953, "SRR6176643", "SRX3287364", "SRS2596838", "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, "DEW065 f2", "GSM2813934", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW065 f2", "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", "GSM2813934", "GSM2813934: DEW065 f2; Danio rerio; RNA Seq", "GSM2813934", 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:GSM2813934", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options:  appendBCtoName", "FC5_DEW-065_S2.R1.fastq.gz FC5_DEW-065_S2.R2.fastq.gz", "fastq fastq", 7093789012.0, 85056439.0, "GSM2813934 r5", null, "A:1558003473;C:1373608340;G:1645617228;T:2515581430;N:978541", null, null, null, null, 1558003473, 1373608340, 1645617228, 2515581430, 978541, "SRX3287364", "SRS2596838", "SRA619743", "GEO", "Harvard University", 2, 0.74182, 0.00129, 0.21487, 0.00114, 0.80154, 0.99965, 0.55567, 0.5, 33, 50, "B", "T", "mate2 technical by mapping diff", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43954, "SRR6176634", "SRX3287363", "SRS2596837", "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, "DEW064 f2", "GSM2813933", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW064 f2", "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", "GSM2813933", "GSM2813933: DEW064 f2; Danio rerio; RNA Seq", "GSM2813933", 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:GSM2813933", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options:  appendBCtoName", "FC1_DEW-064_S7.R1.fastq.gz FC1_DEW-064_S7.R2.fastq.gz", "fastq fastq", 2382079622.0, 28603442.0, "GSM2813933 r1", null, "A:531702920;C:455344015;G:577305407;T:817119830;N:607450", null, null, null, null, 531702920, 455344015, 577305407, 817119830, 607450, "SRX3287363", "SRS2596837", "SRA619743", "GEO", "Harvard University", 2, 0.71162, 0.00613, 0.2075, 0.00587, 0.80081, 0.99945, 0.55002, 0.625, 33, 50, "B", "T", "mate2 technical by mapping diff", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43955, "SRR6176635", "SRX3287363", "SRS2596837", "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, "DEW064 f2", "GSM2813933", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW064 f2", "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", "GSM2813933", "GSM2813933: DEW064 f2; Danio rerio; RNA Seq", "GSM2813933", 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:GSM2813933", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options:  appendBCtoName", "FC2_DEW-064_S7.R1.fastq.gz FC2_DEW-064_S7.R2.fastq.gz", "fastq fastq", 1103522256.0, 13268132.0, "GSM2813933 r2", null, "A:228512922;C:195157576;G:347887797;T:331375946;N:588015", null, null, null, null, 228512922, 195157576, 347887797, 331375946, 588015, "SRX3287363", "SRS2596837", "SRA619743", "GEO", "Harvard University", 2, 0.70781, 0.02127, 0.20824, 0.01963, 0.79776, 0.99922, 0.56118, 0.89265, 33, 50, "B", "T", "mate2 technical by mapping diff", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43956, "SRR6176636", "SRX3287363", "SRS2596837", "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, "DEW064 f2", "GSM2813933", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW064 f2", "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", "GSM2813933", "GSM2813933: DEW064 f2; Danio rerio; RNA Seq", "GSM2813933", 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:GSM2813933", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options:  appendBCtoName", "FC3_DEW-064_S7.R1.fastq.gz FC3_DEW-064_S7.R2.fastq.gz", "fastq fastq", 2482547494.0, 29821327.0, "GSM2813933 r3", null, "A:550735946;C:473925826;G:590960648;T:865736614;N:1188460", null, null, null, null, 550735946, 473925826, 590960648, 865736614, 1188460, "SRX3287363", "SRS2596837", "SRA619743", "GEO", "Harvard University", 2, 0.71704, 0.00423, 0.20978, 0.00397, 0.79618, 0.99945, 0.5645, 0.60526, 33, 50, "B", "T", "mate2 technical by mapping diff", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43957, "SRR6176637", "SRX3287363", "SRS2596837", "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, "DEW064 f2", "GSM2813933", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW064 f2", "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", "GSM2813933", "GSM2813933: DEW064 f2; Danio rerio; RNA Seq", "GSM2813933", 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:GSM2813933", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options:  appendBCtoName", "FC4_DEW-064_S7.R1.fastq.gz FC4_DEW-064_S7.R2.fastq.gz", "fastq fastq", 2339730381.0, 28089056.0, "GSM2813933 r4", null, "A:515317502;C:444338876;G:558021847;T:821750487;N:301669", null, null, null, null, 515317502, 444338876, 558021847, 821750487, 301669, "SRX3287363", "SRS2596837", "SRA619743", "GEO", "Harvard University", 2, 0.71723, 0.00493, 0.20856, 0.00455, 0.79417, 0.999, 0.53427, 0.5, 34, 50, "B", "T", "mate2 technical by mapping diff", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43958, "SRR6176638", "SRX3287363", "SRS2596837", "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, "DEW064 f2", "GSM2813933", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW064 f2", "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", "GSM2813933", "GSM2813933: DEW064 f2; Danio rerio; RNA Seq", "GSM2813933", 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:GSM2813933", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options:  appendBCtoName", "FC5_DEW-064_S1.R1.fastq.gz FC5_DEW-064_S1.R2.fastq.gz", "fastq fastq", 7092166646.0, 85201470.0, "GSM2813933 r5", null, null, null, null, null, null, null, null, null, null, null, "SRX3287363", "SRS2596837", "SRA619743", "GEO", "Harvard University", 2, 0.70151, 0.00121, 0.20646, 0.00113, 0.7917, 0.99971, 0.56301, 0.53333, 34, 50, "B", "T", "mate2 technical by mapping diff", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43959, "SRR6176630", "SRX3287362", "SRS2596836", "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, "DEW063 f1", "GSM2813932", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW063 f1", "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", "GSM2813932", "GSM2813932: DEW063 f1; Danio rerio; RNA Seq", "GSM2813932", 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:GSM2813932", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options:  appendBCtoName", "FC1_DEW-063_S6.R1.fastq.gz FC1_DEW-063_S6.R2.fastq.gz", "fastq fastq", 2703662317.0, 32411788.0, "GSM2813932 r1", null, "A:585487740;C:518930190;G:654356649;T:944203697;N:684041", null, null, null, null, 585487740, 518930190, 654356649, 944203697, 684041, "SRX3287362", "SRS2596836", "SRA619743", "GEO", "Harvard University", 2, 0.73487, 0.005, 0.20711, 0.00478, 0.8057, 0.99953, 0.56192, 0.5, 34, 50, "B", "T", "mate2 technical by mapping diff", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43960, "SRR6176631", "SRX3287362", "SRS2596836", "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, "DEW063 f1", "GSM2813932", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW063 f1", "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", "GSM2813932", "GSM2813932: DEW063 f1; Danio rerio; RNA Seq", "GSM2813932", 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:GSM2813932", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options:  appendBCtoName", "FC2_DEW-063_S6.R1.fastq.gz FC2_DEW-063_S6.R2.fastq.gz", "fastq fastq", 1228068654.0, 14732853.0, "GSM2813932 r2", null, null, null, null, null, null, null, null, null, null, null, "SRX3287362", "SRS2596836", "SRA619743", "GEO", "Harvard University", 2, 0.72354, 0.01516, 0.20566, 0.01388, 0.79793, 0.9992, 0.57243, 0.8492, 33, 50, "B", "T", "mate2 technical by mapping diff", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43961, "SRR6176632", "SRX3287362", "SRS2596836", "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, "DEW063 f1", "GSM2813932", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW063 f1", "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", "GSM2813932", "GSM2813932: DEW063 f1; Danio rerio; RNA Seq", "GSM2813932", 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:GSM2813932", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options:  appendBCtoName", "FC3_DEW-063_S6.R1.fastq.gz FC3_DEW-063_S6.R2.fastq.gz", "fastq fastq", 2865383991.0, 34360219.0, "GSM2813932 r3", null, "A:616333652;C:548965462;G:681196824;T:1017502380;N:1385673", null, null, null, null, 616333652, 548965462, 681196824, 1017502380, 1385673, "SRX3287362", "SRS2596836", "SRA619743", "GEO", "Harvard University", 2, 0.74018, 0.00341, 0.20674, 0.00318, 0.80188, 0.99953, 0.56249, 0.51612, 34, 50, "B", "T", "mate2 technical by mapping diff", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43962, "SRR6176633", "SRX3287362", "SRS2596836", "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, "DEW063 f1", "GSM2813932", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW063 f1", "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", "GSM2813932", "GSM2813932: DEW063 f1; Danio rerio; RNA Seq", "GSM2813932", 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:GSM2813932", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options:  appendBCtoName", "FC4_DEW-063_S6.R1.fastq.gz FC4_DEW-063_S6.R2.fastq.gz", "fastq fastq", 2661201306.0, 31897292.0, "GSM2813932 r4", null, "A:567795696;C:506539944;G:635339481;T:951184460;N:341725", null, null, null, null, 567795696, 506539944, 635339481, 951184460, 341725, "SRX3287362", "SRS2596836", "SRA619743", "GEO", "Harvard University", 2, 0.74623, 0.00451, 0.20997, 0.00422, 0.79949, 0.99928, 0.56605, 0.4375, 34, 50, "B", "T", "mate2 technical by mapping diff", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43963, "SRR6176626", "SRX3287361", "SRS2596835", "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, "DEW062 f1", "GSM2813931", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW062 f1", "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", "GSM2813931", "GSM2813931: DEW062 f1; Danio rerio; RNA Seq", "GSM2813931", 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:GSM2813931", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options:  appendBCtoName", "FC1_DEW-062_S5.R1.fastq.gz FC1_DEW-062_S5.R2.fastq.gz", "fastq fastq", 2694116042.0, 32276313.0, "GSM2813931 r1", null, "A:589473486;C:513444615;G:648306227;T:942198524;N:693190", null, null, null, null, 589473486, 513444615, 648306227, 942198524, 693190, "SRX3287361", "SRS2596835", "SRA619743", "GEO", "Harvard University", 2, 0.75006, 0.00665, 0.22251, 0.00642, 0.79776, 0.99953, 0.56954, 0.53571, 33, 50, "B", "T", "mate2 technical by mapping diff", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43964, "SRR6176627", "SRX3287361", "SRS2596835", "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, "DEW062 f1", "GSM2813931", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW062 f1", "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", "GSM2813931", "GSM2813931: DEW062 f1; Danio rerio; RNA Seq", "GSM2813931", 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:GSM2813931", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options:  appendBCtoName", "FC2_DEW-062_S5.R1.fastq.gz FC2_DEW-062_S5.R2.fastq.gz", "fastq fastq", 1221053333.0, 14634722.0, "GSM2813931 r2", null, "A:246914186;C:214804950;G:383568470;T:375108039;N:657688", null, null, null, null, 246914186, 214804950, 383568470, 375108039, 657688, "SRX3287361", "SRS2596835", "SRA619743", "GEO", "Harvard University", 2, 0.7539, 0.0214, 0.22301, 0.02015, 0.79681, 0.99945, 0.56759, 0.82089, 34, 50, "B", "T", "mate2 technical by mapping diff", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43965, "SRR6176628", "SRX3287361", "SRS2596835", "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, "DEW062 f1", "GSM2813931", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW062 f1", "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", "GSM2813931", "GSM2813931: DEW062 f1; Danio rerio; RNA Seq", "GSM2813931", 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:GSM2813931", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options:  appendBCtoName", "FC3_DEW-062_S5.R1.fastq.gz FC3_DEW-062_S5.R2.fastq.gz", "fastq fastq", 2732077263.0, 32737784.0, "GSM2813931 r3", null, "A:593509115;C:520050361;G:645630165;T:971575716;N:1311906", null, null, null, null, 593509115, 520050361, 645630165, 971575716, 1311906, "SRX3287361", "SRS2596835", "SRA619743", "GEO", "Harvard University", 2, 0.75396, 0.00435, 0.22336, 0.00415, 0.79257, 0.99957, 0.566, 0.62068, 34, 50, "B", "T", "mate2 technical by mapping diff", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43966, "SRR6176629", "SRX3287361", "SRS2596835", "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, "DEW062 f1", "GSM2813931", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW062 f1", "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", "GSM2813931", "GSM2813931: DEW062 f1; Danio rerio; RNA Seq", "GSM2813931", 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:GSM2813931", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options:  appendBCtoName", "FC4_DEW-062_S5.R1.fastq.gz FC4_DEW-062_S5.R2.fastq.gz", "fastq fastq", 2513049040.0, 30103618.0, "GSM2813931 r4", null, "A:540807825;C:475246041;G:596916568;T:899752561;N:326045", null, null, null, null, 540807825, 475246041, 596916568, 899752561, 326045, "SRX3287361", "SRS2596835", "SRA619743", "GEO", "Harvard University", 2, 0.7577, 0.00581, 0.22002, 0.00546, 0.79131, 0.99912, 0.5594, 0.69491, 33, 50, "B", "T", "mate2 technical by mapping diff", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43967, "SRR6176622", "SRX3287360", "SRS2596834", "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, "DEW061 f1", "GSM2813930", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW061 f1", "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", "GSM2813930", "GSM2813930: DEW061 f1; Danio rerio; RNA Seq", "GSM2813930", 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:GSM2813930", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options:  appendBCtoName", "FC1_DEW-061_S4.R1.fastq.gz FC1_DEW-061_S4.R2.fastq.gz", "fastq fastq", 3154139976.0, 37801014.0, "GSM2813930 r1", null, "A:693516487;C:600830347;G:760784710;T:1098206077;N:802355", null, null, null, null, 693516487, 600830347, 760784710, 1098206077, 802355, "SRX3287360", "SRS2596834", "SRA619743", "GEO", "Harvard University", 2, 0.74925, 0.00596, 0.22537, 0.00573, 0.79271, 0.99961, 0.55809, 0.5, 34, 50, "B", "T", "mate2 technical by mapping diff", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43968, "SRR6176623", "SRX3287360", "SRS2596834", "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, "DEW061 f1", "GSM2813930", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW061 f1", "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", "GSM2813930", "GSM2813930: DEW061 f1; Danio rerio; RNA Seq", "GSM2813930", 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:GSM2813930", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options:  appendBCtoName", "FC2_DEW-061_S4.R1.fastq.gz FC2_DEW-061_S4.R2.fastq.gz", "fastq fastq", 1440125590.0, 17268172.0, "GSM2813930 r2", null, "A:292214170;C:252368952;G:455430577;T:439332662;N:779229", null, null, null, null, 292214170, 252368952, 455430577, 439332662, 779229, "SRX3287360", "SRS2596834", "SRA619743", "GEO", "Harvard University", 2, 0.75669, 0.02314, 0.22842, 0.02167, 0.79393, 0.99943, 0.55096, 0.91666, 34, 50, "B", "T", "mate2 technical by mapping diff", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43969, "SRR6176624", "SRX3287360", "SRS2596834", "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, "DEW061 f1", "GSM2813930", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW061 f1", "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", "GSM2813930", "GSM2813930: DEW061 f1; Danio rerio; RNA Seq", "GSM2813930", 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:GSM2813930", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options:  appendBCtoName", "FC3_DEW-061_S4.R1.fastq.gz FC3_DEW-061_S4.R2.fastq.gz", "fastq fastq", 3242023471.0, 38863564.0, "GSM2813930 r3", null, "A:708050133;C:616691319;G:767462847;T:1148237613;N:1581559", null, null, null, null, 708050133, 616691319, 767462847, 1148237613, 1581559, "SRX3287360", "SRS2596834", "SRA619743", "GEO", "Harvard University", 2, 0.75359, 0.00376, 0.22736, 0.00349, 0.79123, 0.99939, 0.55827, 0.54054, 34, 50, "B", "T", "mate2 technical by mapping diff", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43970, "SRR6176625", "SRX3287360", "SRS2596834", "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, "DEW061 f1", "GSM2813930", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW061 f1", "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", "GSM2813930", "GSM2813930: DEW061 f1; Danio rerio; RNA Seq", "GSM2813930", 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:GSM2813930", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options:  appendBCtoName", "FC4_DEW-061_S4.R1.fastq.gz FC4_DEW-061_S4.R2.fastq.gz", "fastq fastq", 2998511059.0, 35932183.0, "GSM2813930 r4", null, "A:648015203;C:566278950;G:715347339;T:1068484671;N:384896", null, null, null, null, 648015203, 566278950, 715347339, 1068484671, 384896, "SRX3287360", "SRS2596834", "SRA619743", "GEO", "Harvard University", 2, 0.75921, 0.00595, 0.22572, 0.00561, 0.78782, 0.9991, 0.5551, 0.54545, 33, 50, "B", "T", "mate2 technical by mapping diff", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43971, "SRR6176618", "SRX3287359", "SRS2596833", "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, "DEW060 f1", "GSM2813929", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW060 f1", "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", "GSM2813929", "GSM2813929: DEW060 f1; Danio rerio; RNA Seq", "GSM2813929", 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:GSM2813929", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options:  appendBCtoName", "FC1_DEW-060_S3.R1.fastq.gz FC1_DEW-060_S3.R2.fastq.gz", "fastq fastq", 2408131518.0, 28860483.0, "GSM2813929 r1", null, "A:529387121;C:457366350;G:579506465;T:841263578;N:608004", null, null, null, null, 529387121, 457366350, 579506465, 841263578, 608004, "SRX3287359", "SRS2596833", "SRA619743", "GEO", "Harvard University", 2, 0.74623, 0.00668, 0.22553, 0.00648, 0.79776, 0.99959, 0.56194, 0.33333, 34, 50, "B", "T", "mate2 technical by mapping diff", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43972, "SRR6176619", "SRX3287359", "SRS2596833", "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, "DEW060 f1", "GSM2813929", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW060 f1", "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", "GSM2813929", "GSM2813929: DEW060 f1; Danio rerio; RNA Seq", "GSM2813929", 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:GSM2813929", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options:  appendBCtoName", "FC2_DEW-060_S3.R1.fastq.gz FC2_DEW-060_S3.R2.fastq.gz", "fastq fastq", 1150468859.0, 13794506.0, "GSM2813929 r2", null, "A:233434027;C:201107426;G:362797690;T:352510992;N:618724", null, null, null, null, 233434027, 201107426, 362797690, 352510992, 618724, "SRX3287359", "SRS2596833", "SRA619743", "GEO", "Harvard University", 2, 0.75233, 0.02671, 0.22646, 0.0248, 0.79454, 0.99955, 0.55277, 0.91351, 34, 50, "B", "T", "mate2 technical by mapping diff", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43973, "SRR6176620", "SRX3287359", "SRS2596833", "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, "DEW060 f1", "GSM2813929", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW060 f1", "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", "GSM2813929", "GSM2813929: DEW060 f1; Danio rerio; RNA Seq", "GSM2813929", 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:GSM2813929", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options:  appendBCtoName", "FC3_DEW-060_S3.R1.fastq.gz FC3_DEW-060_S3.R2.fastq.gz", "fastq fastq", 2490214284.0, 29850978.0, "GSM2813929 r3", null, "A:542990500;C:472486099;G:588451265;T:885110481;N:1175939", null, null, null, null, 542990500, 472486099, 588451265, 885110481, 1175939, "SRX3287359", "SRS2596833", "SRA619743", "GEO", "Harvard University", 2, 0.75217, 0.00417, 0.22495, 0.00392, 0.79371, 0.99945, 0.55373, 0.57575, 33, 50, "B", "T", "mate2 technical by mapping diff", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43974, "SRR6176621", "SRX3287359", "SRS2596833", "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, "DEW060 f1", "GSM2813929", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW060 f1", "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", "GSM2813929", "GSM2813929: DEW060 f1; Danio rerio; RNA Seq", "GSM2813929", 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:GSM2813929", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options:  appendBCtoName", "FC4_DEW-060_S3.R1.fastq.gz FC4_DEW-060_S3.R2.fastq.gz", "fastq fastq", 2335848886.0, 27991106.0, "GSM2813929 r4", null, "A:504358369;C:440180845;G:555523061;T:835493929;N:292682", null, null, null, null, 504358369, 440180845, 555523061, 835493929, 292682, "SRX3287359", "SRS2596833", "SRA619743", "GEO", "Harvard University", 2, 0.75612, 0.00625, 0.22331, 0.00595, 0.7907, 0.99933, 0.55242, 0.55555, 34, 50, "B", "T", "mate2 technical by mapping diff", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43975, "SRR6176614", "SRX3287358", "SRS2596832", "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, "DEW059 f1", "GSM2813928", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW059 f1", "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", "GSM2813928", "GSM2813928: DEW059 f1; Danio rerio; RNA Seq", "GSM2813928", 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:GSM2813928", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options:  appendBCtoName", "FC1_DEW-059_S2.R1.fastq.gz FC1_DEW-059_S2.R2.fastq.gz", "fastq fastq", 2468427036.0, 29617911.0, "GSM2813928 r1", null, "A:542065912;C:474767208;G:599126438;T:851830072;N:637406", null, null, null, null, 542065912, 474767208, 599126438, 851830072, 637406, "SRX3287358", "SRS2596832", "SRA619743", "GEO", "Harvard University", 2, 0.72834, 0.00622, 0.21554, 0.00591, 0.79888, 0.99941, 0.54791, 0.62162, 33, 49, "B", "T", "mate2 technical by mapping diff", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43976, "SRR6176615", "SRX3287358", "SRS2596832", "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, "DEW059 f1", "GSM2813928", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW059 f1", "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", "GSM2813928", "GSM2813928: DEW059 f1; Danio rerio; RNA Seq", "GSM2813928", 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:GSM2813928", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options:  appendBCtoName", "FC2_DEW-059_S2.R1.fastq.gz FC2_DEW-059_S2.R2.fastq.gz", "fastq fastq", 1116808176.0, 13413942.0, "GSM2813928 r2", null, "A:227203028;C:198647634;G:352135639;T:338217127;N:604748", null, null, null, null, 227203028, 198647634, 352135639, 338217127, 604748, "SRX3287358", "SRS2596832", "SRA619743", "GEO", "Harvard University", 2, 0.72547, 0.02085, 0.21465, 0.01937, 0.79878, 0.99949, 0.55459, 0.91719, 28, 49, "T", "B", "sc-like readlen", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43977, "SRR6176616", "SRX3287358", "SRS2596832", "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, "DEW059 f1", "GSM2813928", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW059 f1", "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", "GSM2813928", "GSM2813928: DEW059 f1; Danio rerio; RNA Seq", "GSM2813928", 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:GSM2813928", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options:  appendBCtoName", "FC3_DEW-059_S2.R1.fastq.gz FC3_DEW-059_S2.R2.fastq.gz", "fastq fastq", 2433240009.0, 29205364.0, "GSM2813928 r3", null, "A:530808152;C:467393483;G:580237225;T:853631720;N:1169429", null, null, null, null, 530808152, 467393483, 580237225, 853631720, 1169429, "SRX3287358", "SRS2596832", "SRA619743", "GEO", "Harvard University", 2, 0.73343, 0.00393, 0.21608, 0.00374, 0.79446, 0.99961, 0.55546, 0.66666, 34, 50, "B", "T", "mate2 technical by mapping diff", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43978, "SRR6176617", "SRX3287358", "SRS2596832", "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, "DEW059 f1", "GSM2813928", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW059 f1", "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", "GSM2813928", "GSM2813928: DEW059 f1; Danio rerio; RNA Seq", "GSM2813928", 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:GSM2813928", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options:  appendBCtoName", "FC4_DEW-059_S2.R1.fastq.gz FC4_DEW-059_S2.R2.fastq.gz", "fastq fastq", 2254112176.0, 27041760.0, "GSM2813928 r4", null, "A:488078877;C:430617586;G:538610970;T:796514276;N:290467", null, null, null, null, 488078877, 430617586, 538610970, 796514276, 290467, "SRX3287358", "SRS2596832", "SRA619743", "GEO", "Harvard University", 2, 0.73283, 0.00521, 0.21537, 0.0049, 0.79375, 0.9991, 0.5546, 0.56603, 34, 50, "B", "T", "mate2 technical by mapping diff", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43979, "SRR6176610", "SRX3287357", "SRS2596831", "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, "DEW058 f1", "GSM2813927", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW058 f1", "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", "GSM2813927", "GSM2813927: DEW058 f1; Danio rerio; RNA Seq", "GSM2813927", 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:GSM2813927", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options:  appendBCtoName", "FC1_DEW-058_S1.R1.fastq.gz FC1_DEW-058_S1.R2.fastq.gz", "fastq fastq", 2403771793.0, 28792952.0, "GSM2813927 r1", null, "A:517810269;C:461480878;G:579161807;T:844718642;N:600197", null, null, null, null, 517810269, 461480878, 579161807, 844718642, 600197, "SRX3287357", "SRS2596831", "SRA619743", "GEO", "Harvard University", 2, 0.75451, 0.00612, 0.2178, 0.00596, 0.79953, 0.99963, 0.5419, 0.5, 34, 49, "B", "T", "mate2 technical by mapping diff", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "indrops", null, "United States", "2017-10-16", "Larval", "Larval", "Brain", "Nervous System"], [43980, "SRR6176611", "SRX3287357", "SRS2596831", "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, "DEW058 f1", "GSM2813927", null, "source name:zebrafish brain|tissue:brain|developmental stage:23 25dpf", "DEW058 f1", "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", "GSM2813927", "GSM2813927: DEW058 f1; Danio rerio; RNA Seq", "GSM2813927", 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:GSM2813927", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP120009", null, "loader:fastq load.py|options:  appendBCtoName", "FC2_DEW-058_S1.R1.fastq.gz FC2_DEW-058_S1.R2.fastq.gz", "fastq fastq", 1112151214.0, 13326194.0, "GSM2813927 r2", null, null, null, null, null, null, null, null, null, null, null, "SRX3287357", "SRS2596831", "SRA619743", "GEO", "Harvard University", 2, 0.75157, 0.01674, 0.22743, 0.01541, 0.79145, 0.99924, 0.55163, 0.86311, 34, 49, "B", "T", "mate2 technical by mapping diff", "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": 116, "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, 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