{"database": "metadata", "table": "run_metadata", "is_view": false, "human_description_en": "where experiment.library_selection = \"other\", experiment.library_strategy = \"OTHER\" and tissue_curation = \"Whole Organism\"", "rows": [[25295, "SRR25764045", "SRX21486723", "SRS18719024", "SRP457105", "PRJNA1009809", "Dynamics of the zebrafish tRNAome during the maternal to zygotic transition [Ribo Seq]", "GSE241753", "Other", "Time course analysis of tRNA abundance during zebrafish early embryonic development. Overall design: Wild type TLAB strain zebrafish embryos were grown in standard housing conditions.Unfertilized eggs 0 hpf were collected or embryos were staged and collected at consecutive developmental time points namelyat the 256 cell 2.5 hpf 1000 cell 3 hpf sphere 4 hpf shield 6 hpf and bud 10 hpf stages. Eggs and embryos were either flash frozen in liquid nitrogen or immediately processed. Samples were used for western blotting analysis polysome profiling ribosome profiling or mRNA and tRNA sequencing for investigating the regulation of tRNA gene expression and translational status during early zebrafish embryogenesis.", "parent bioproject:PRJNA1009800", "pubmed:39402326", null, "WT bud 10 hpf Ribo seq rep1", "GSM7734770", null, "source name:Gastrula|strain:TLAB strain|tissue:Gastrula|developmental stage:Bud 10 hpf|treatment:100 \u00b5g/ml CHX 100 \u00b5g/ml TIG|geo loc name:missing|collection date:missing", "WT bud 10 hpf Ribo seq rep1", "We identified the A site location in each mapped read with Scikit ribo [Fang et al. 2018] which uses a random forest with recursive feature selection and a generalized linear model for accurate A site prediction based on matched ribosome profiling and RNA Seq datasets. Kallisto 0.44.0 with parameters b 100 single l 180 s 20 t 40 was used to quantify transcript abundances in Transcripts Per Million TPM from RNA Seq data based on the reference set of MANE annotated transcripts see Codon usage analysis for description of this annotation. To avoid memory errors due to the large size of the human genome and the presence of multiple transcript isoforms all RNAfold dependencies in Scikit ribo were omitted and the index was built separately for each chromosome. To make the hg38 GTF compatible with Scikit ribo transcript/UTR annotations were removed. For each transcript the start codon in the first exon and the stop codon in the last exon were adjusted to represent transcript start and end coordinates taking into account the gene strand. To estimate codon dwell times short 20 23 nt and long 28 33 nt ribosome footprints were analyzed separately. rRNA filtered reads were aligned to GRCz11.108 using STAR v2.6.1c [Dobin et al. 2013] the following options: outFilterMultimapNmax 1 seedSearchStartLmax 15 outSAMtype BAM SortedByCoordinate outFilterMismatchNmax 2 alignEndsType EndToEnd quantMode TranscriptomeSAM outSAMattributes NH HI AS nM NM MD We identified the A site location in each mapped read with Scikit ribo [Fang et al. 2018] which uses a random forest with recursive feature selection and a generalized linear model for accurate A site prediction based on matched ribosome profiling and RNA Seq datasets. Kallisto 0.44.0 with parameters b 100 single l 180 s 20 t 40 was used to quantify transcript abundances in Transcripts Per Million TPM from RNA Seq data based on the reference set of MANE annotated transcripts see Codon usage analysis for description of this annotation. To avoid memory errors due to the large size of the human genome and the presence of multiple transcript isoforms all RNAfold dependencies in Scikit ribo were omitted and the index was built separately for each chromosome. To make the GRCz11 GTF compatible with Scikit ribo transcript/UTR annotations were removed. For each transcript the start codon in the first exon and the stop codon in the last exon were adjusted to represent transcript start and end coordinates taking into account the gene strand. To estimate codon dwell times we utilised ribosome footprints with length 30 34 nt. Assembly: GRCz11 Supplementary files format and content: csv; codon dwell times from scikit ribo for all samples Supplementary files format and content: csv; transcript per million TPM counts per transcript in MANE annotation Library strategy: Ribo Seq", "Gastrula", "unperturbed growth conditions in E3 medium for zebrafish embryos.", "200 whole embryos were flash frozen in liquid nitrogen and subsequently lysed in footprint lysis buffer containing 100 \u00b5g/ml CHX and 100 \u00b5g/ml TIG 0.1% NP 40 10 \u00b5g/ml aprotinin 20 \u00b5M leupeptin 2.5 \u00b5M pepstatin A 0.5 mM AEBSF and 1x Phosphatase Inhibitor Cocktail. Samples were vortexed vigorously triturated through a 26G gauge needle and spun down for 7 minutes at 16 000xg/ 4\u00b0C. Supernatant was transferred to a new tube. 20 \u00b5g RNA in 200 \u00b5l polysome lysis buffer were digested with 50 U RNase I for 45 minutes at 2 000 rpm/22\u00b0C. post incubation on ice for 5 minutes extracts were pre cleared by centrifugation for 5 minutes at 3 000 g/ 4\u00b0C. Ribosomes were pelleted through 3 ml of a sucrose cushion 1 M sucrose 20 mM Tris pH=8.0 140 mM KCl 5 mM MgCl2 1 mM DTT by spinning the layered solutions in the Type 70 Ti rotor for 120 minutes at 50 000 rpm/ 4\u00b0C. Ribosome pellets were rinsed once dissolved in 200 \u00b5l drug free polysome lysis buffer and incubated with 200 U hiPSC or 300 U NPC RNase I for 45 minutes at 2 000 rpm/22\u00b0C. Ribosome footprint libraries were prepared essentially as described McGlincy and Ingolia 2017; Wu 2019 with minor modifications. RNase I digestion was stopped by addition of 100 U Superase In and extracts were loaded on a sucrose cushion. The pellet was dissolved in 400 \u00b5l LiDS/LET lysis buffer and RNA was extracted with the acid phenol protocol. Fragments in the range of 19 to 32 nucleotides were isolated by gel size selection with T4 PNK and ligated to pre adenylated adapters containing 5 random nucleotides at their 5\u2019 ends McGlinzy 2017 with T4 RNA Ligase 2 truncated KQ. Adapter ligated RNA was subjected to rRNA depletion using the Ribo Seq riboPOOL h/m/r depletion kit siTOOLs for CHX only samples and legacy RiboZero Gold kit Illumina for CHX+TIG samples The rRNA depleted footprints were reverse transcribed with Protoscript II and cDNA was circularized with recombinant TS2126 RNA ligase 1 commercially available as CircLigase. Libraries were constructed from circularized cDNA with KAPA HiFi DNA Polymerase and single end sequencing was performed on a NextSeq 500 platform Illumina.", "Embryos were grown in standard housing conditions namely28\u00b0C at a 14/10 hour light/dark cycle.", "strain:TLAB strain|tissue:Gastrula|developmental stage:Bud 10 hpf|treatment:100 \u00b5g/ml CHX 100 \u00b5g/ml TIG", "GSM7734770", "GSM7734770: WT bud 10 hpf Ribo seq rep1; Danio rerio; OTHER", "GSM7734770 r1", "GSM7734770", "1", "200 whole embryos were flash frozen in liquid nitrogen and subsequently lysed in footprint lysis buffer containing 100 \u00b5g/ml CHX and 100 \u00b5g/ml TIG 0.1% NP 40 10 \u00b5g/ml aprotinin 20 \u00b5M leupeptin 2.5 \u00b5M pepstatin A 0.5 mM AEBSF and 1x Phosphatase Inhibitor Cocktail. Samples were vortexed vigorously triturated through a 26G gauge needle and spun down for 7 minutes at 16 000xg/ 4\u00b0C. Supernatant was transferred to a new tube. 20 \u00b5g RNA in 200 \u00b5l polysome lysis buffer were digested with 50 U RNase I for 45 minutes at 2 000 rpm/22\u00b0C. post incubation on ice for 5 minutes extracts were pre cleared by centrifugation for 5 minutes at 3 000 g/ 4\u00b0C. Ribosomes were pelleted through 3 ml of a sucrose cushion 1 M sucrose 20 mM Tris pH=8.0 140 mM KCl 5 mM MgCl2 1 mM DTT by spinning the layered solutions in the Type 70 Ti rotor for 120 minutes at 50 000 rpm/ 4\u00b0C. Ribosome pellets were rinsed once dissolved in 200 \u00b5l drug free polysome lysis buffer and incubated with 200 U hiPSC or 300 U NPC RNase I for 45 minutes at 2 000 rpm/22\u00b0C. Ribosome footprint libraries were prepared essentially as described McGlincy and Ingolia 2017; Wu 2019 with minor modifications. RNase I digestion was stopped by addition of 100 U Superase In and extracts were loaded on a sucrose cushion. The pellet was dissolved in 400 \u00b5l LiDS/LET lysis buffer and RNA was extracted with the acid phenol protocol. Fragments in the range of 19 to 32 nucleotides were isolated by gel size selection with T4 PNK and ligated to pre adenylated adapters containing 5 random nucleotides at their five prime ends McGlinzy 2017 with T4 RNA Ligase 2 truncated KQ. Adapter ligated RNA was subjected to rRNA depletion using the Ribo Seq riboPOOL h/m/r depletion kit siTOOLs for CHX only samples and legacy RiboZero Gold kit Illumina for CHX+TIG samples The rRNA depleted footprints were reverse transcribed with Protoscript II and cDNA was circularized with recombinant TS2126 RNA ligase 1 commercially available as CircLigase. Libraries were constructed from circularized cDNA with KAPA HiFi DNA Polymerase and single end sequencing was performed on a NextSeq 500 platform Illumina.", null, "OTHER", "TRANSCRIPTOMIC", "other", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP457105", null, null, "WT_ribo_bud_1.fastq.gz", "fastq", 1476985703.0, 55745002.0, "GSM7734770 r1", "0:26.50", "A:266369385;C:466461341;G:473469631;T:270671671;N:13675", 26, null, null, null, 266369385, 466461341, 473469631, 270671671, 13675, "SRX21486723", "SRS18719024", "SRA1700409", "Mechanisms of Protein Biogenesis, Max Planck Institute for Biochemistry", "Max Planck Institute of Biochemistry", 1, 0.773, null, 0.14121, null, 0.82242, null, 0.78464, null, 30, null, "B", null, "usable mapping rate", "illumina", "nextseq", "5prime", "rrna_depletion", "ribozero", "bulk", "unknown", "unknown", null, "Germany", "2023-08-28", "Gastrula", "Embryo", "Whole Organism", "All anatomical structures"], [25296, "SRR25764046", "SRX21486722", "SRS18719023", "SRP457105", "PRJNA1009809", "Dynamics of the zebrafish tRNAome during the maternal to zygotic transition [Ribo Seq]", "GSE241753", "Other", "Time course analysis of tRNA abundance during zebrafish early embryonic development. Overall design: Wild type TLAB strain zebrafish embryos were grown in standard housing conditions.Unfertilized eggs 0 hpf were collected or embryos were staged and collected at consecutive developmental time points namelyat the 256 cell 2.5 hpf 1000 cell 3 hpf sphere 4 hpf shield 6 hpf and bud 10 hpf stages. Eggs and embryos were either flash frozen in liquid nitrogen or immediately processed. Samples were used for western blotting analysis polysome profiling ribosome profiling or mRNA and tRNA sequencing for investigating the regulation of tRNA gene expression and translational status during early zebrafish embryogenesis.", "parent bioproject:PRJNA1009800", "pubmed:39402326", null, "WT sphere 4 hpf Ribo seq rep1", "GSM7734769", null, "source name:Blastula|strain:TLAB strain|tissue:Blastula|developmental stage:Sphere 4 hpf|treatment:100 \u00b5g/ml CHX 100 \u00b5g/ml TIG|geo loc name:missing|collection date:missing", "WT sphere 4 hpf Ribo seq rep1", "We identified the A site location in each mapped read with Scikit ribo [Fang et al. 2018] which uses a random forest with recursive feature selection and a generalized linear model for accurate A site prediction based on matched ribosome profiling and RNA Seq datasets. Kallisto 0.44.0 with parameters b 100 single l 180 s 20 t 40 was used to quantify transcript abundances in Transcripts Per Million TPM from RNA Seq data based on the reference set of MANE annotated transcripts see Codon usage analysis for description of this annotation. To avoid memory errors due to the large size of the human genome and the presence of multiple transcript isoforms all RNAfold dependencies in Scikit ribo were omitted and the index was built separately for each chromosome. To make the hg38 GTF compatible with Scikit ribo transcript/UTR annotations were removed. For each transcript the start codon in the first exon and the stop codon in the last exon were adjusted to represent transcript start and end coordinates taking into account the gene strand. To estimate codon dwell times short 20 23 nt and long 28 33 nt ribosome footprints were analyzed separately. rRNA filtered reads were aligned to GRCz11.108 using STAR v2.6.1c [Dobin et al. 2013] the following options: outFilterMultimapNmax 1 seedSearchStartLmax 15 outSAMtype BAM SortedByCoordinate outFilterMismatchNmax 2 alignEndsType EndToEnd quantMode TranscriptomeSAM outSAMattributes NH HI AS nM NM MD We identified the A site location in each mapped read with Scikit ribo [Fang et al. 2018] which uses a random forest with recursive feature selection and a generalized linear model for accurate A site prediction based on matched ribosome profiling and RNA Seq datasets. Kallisto 0.44.0 with parameters b 100 single l 180 s 20 t 40 was used to quantify transcript abundances in Transcripts Per Million TPM from RNA Seq data based on the reference set of MANE annotated transcripts see Codon usage analysis for description of this annotation. To avoid memory errors due to the large size of the human genome and the presence of multiple transcript isoforms all RNAfold dependencies in Scikit ribo were omitted and the index was built separately for each chromosome. To make the GRCz11 GTF compatible with Scikit ribo transcript/UTR annotations were removed. For each transcript the start codon in the first exon and the stop codon in the last exon were adjusted to represent transcript start and end coordinates taking into account the gene strand. To estimate codon dwell times we utilised ribosome footprints with length 30 34 nt. Assembly: GRCz11 Supplementary files format and content: csv; codon dwell times from scikit ribo for all samples Supplementary files format and content: csv; transcript per million TPM counts per transcript in MANE annotation Library strategy: Ribo Seq", "Blastula", "unperturbed growth conditions in E3 medium for zebrafish embryos.", "200 whole embryos were flash frozen in liquid nitrogen and subsequently lysed in footprint lysis buffer containing 100 \u00b5g/ml CHX and 100 \u00b5g/ml TIG 0.1% NP 40 10 \u00b5g/ml aprotinin 20 \u00b5M leupeptin 2.5 \u00b5M pepstatin A 0.5 mM AEBSF and 1x Phosphatase Inhibitor Cocktail. Samples were vortexed vigorously triturated through a 26G gauge needle and spun down for 7 minutes at 16 000xg/ 4\u00b0C. Supernatant was transferred to a new tube. 20 \u00b5g RNA in 200 \u00b5l polysome lysis buffer were digested with 50 U RNase I for 45 minutes at 2 000 rpm/22\u00b0C. post incubation on ice for 5 minutes extracts were pre cleared by centrifugation for 5 minutes at 3 000 g/ 4\u00b0C. Ribosomes were pelleted through 3 ml of a sucrose cushion 1 M sucrose 20 mM Tris pH=8.0 140 mM KCl 5 mM MgCl2 1 mM DTT by spinning the layered solutions in the Type 70 Ti rotor for 120 minutes at 50 000 rpm/ 4\u00b0C. Ribosome pellets were rinsed once dissolved in 200 \u00b5l drug free polysome lysis buffer and incubated with 200 U hiPSC or 300 U NPC RNase I for 45 minutes at 2 000 rpm/22\u00b0C. Ribosome footprint libraries were prepared essentially as described McGlincy and Ingolia 2017; Wu 2019 with minor modifications. RNase I digestion was stopped by addition of 100 U Superase In and extracts were loaded on a sucrose cushion. The pellet was dissolved in 400 \u00b5l LiDS/LET lysis buffer and RNA was extracted with the acid phenol protocol. Fragments in the range of 19 to 32 nucleotides were isolated by gel size selection with T4 PNK and ligated to pre adenylated adapters containing 5 random nucleotides at their 5\u2019 ends McGlinzy 2017 with T4 RNA Ligase 2 truncated KQ. Adapter ligated RNA was subjected to rRNA depletion using the Ribo Seq riboPOOL h/m/r depletion kit siTOOLs for CHX only samples and legacy RiboZero Gold kit Illumina for CHX+TIG samples The rRNA depleted footprints were reverse transcribed with Protoscript II and cDNA was circularized with recombinant TS2126 RNA ligase 1 commercially available as CircLigase. Libraries were constructed from circularized cDNA with KAPA HiFi DNA Polymerase and single end sequencing was performed on a NextSeq 500 platform Illumina.", "Embryos were grown in standard housing conditions namely28\u00b0C at a 14/10 hour light/dark cycle.", "strain:TLAB strain|tissue:Blastula|developmental stage:Sphere 4 hpf|treatment:100 \u00b5g/ml CHX 100 \u00b5g/ml TIG", "GSM7734769", "GSM7734769: WT sphere 4 hpf Ribo seq rep1; Danio rerio; OTHER", "GSM7734769 r1", "GSM7734769", "1", "200 whole embryos were flash frozen in liquid nitrogen and subsequently lysed in footprint lysis buffer containing 100 \u00b5g/ml CHX and 100 \u00b5g/ml TIG 0.1% NP 40 10 \u00b5g/ml aprotinin 20 \u00b5M leupeptin 2.5 \u00b5M pepstatin A 0.5 mM AEBSF and 1x Phosphatase Inhibitor Cocktail. Samples were vortexed vigorously triturated through a 26G gauge needle and spun down for 7 minutes at 16 000xg/ 4\u00b0C. Supernatant was transferred to a new tube. 20 \u00b5g RNA in 200 \u00b5l polysome lysis buffer were digested with 50 U RNase I for 45 minutes at 2 000 rpm/22\u00b0C. post incubation on ice for 5 minutes extracts were pre cleared by centrifugation for 5 minutes at 3 000 g/ 4\u00b0C. Ribosomes were pelleted through 3 ml of a sucrose cushion 1 M sucrose 20 mM Tris pH=8.0 140 mM KCl 5 mM MgCl2 1 mM DTT by spinning the layered solutions in the Type 70 Ti rotor for 120 minutes at 50 000 rpm/ 4\u00b0C. Ribosome pellets were rinsed once dissolved in 200 \u00b5l drug free polysome lysis buffer and incubated with 200 U hiPSC or 300 U NPC RNase I for 45 minutes at 2 000 rpm/22\u00b0C. Ribosome footprint libraries were prepared essentially as described McGlincy and Ingolia 2017; Wu 2019 with minor modifications. RNase I digestion was stopped by addition of 100 U Superase In and extracts were loaded on a sucrose cushion. The pellet was dissolved in 400 \u00b5l LiDS/LET lysis buffer and RNA was extracted with the acid phenol protocol. Fragments in the range of 19 to 32 nucleotides were isolated by gel size selection with T4 PNK and ligated to pre adenylated adapters containing 5 random nucleotides at their five prime ends McGlinzy 2017 with T4 RNA Ligase 2 truncated KQ. Adapter ligated RNA was subjected to rRNA depletion using the Ribo Seq riboPOOL h/m/r depletion kit siTOOLs for CHX only samples and legacy RiboZero Gold kit Illumina for CHX+TIG samples The rRNA depleted footprints were reverse transcribed with Protoscript II and cDNA was circularized with recombinant TS2126 RNA ligase 1 commercially available as CircLigase. Libraries were constructed from circularized cDNA with KAPA HiFi DNA Polymerase and single end sequencing was performed on a NextSeq 500 platform Illumina.", null, "OTHER", "TRANSCRIPTOMIC", "other", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP457105", null, null, "WT_ribo_sphere_1.fastq.gz", "fastq", 1336027898.0, 47907512.0, "GSM7734769 r1", "0:27.89", "A:229038284;C:439455963;G:429428292;T:238088512;N:16847", 27, null, null, null, 229038284, 439455963, 429428292, 238088512, 16847, "SRX21486722", "SRS18719023", "SRA1700409", "Mechanisms of Protein Biogenesis, Max Planck Institute for Biochemistry", "Max Planck Institute of Biochemistry", 1, 0.85867, null, 0.20418, null, 0.8776, null, 0.79481, null, 24, null, "B", null, "usable mapping rate", "illumina", "nextseq", "5prime", "rrna_depletion", "ribozero", "bulk", "unknown", "unknown", null, "Germany", "2023-08-28", "Blastula", "Embryo", "Whole Organism", "All anatomical structures"], [31943, "SRR28790250", "SRX24354554", "SRS21112322", "SRP503769", "PRJNA1104180", "Double stranded RNA triggers a distinct integrated stress response in the early embryo [RNA seq Ribo seq]", "GSE265771", "Other", "Double stranded RNA dsRNA is associated with virus infections and is present as by products during the transcription of synthetic mRNA which has been widely used in gene gain of function studies and serves as a core component in emerging mRNA based therapies1 4. The presence of dsRNA in host cells induces an integrated stress response that functions to prevent virus replication and infection5 6. Unlike differentiated cells undifferentiated cells adopt a distinct defense strategy against RNA virus infection7 but the mechanism is unclear. We show a previously unidentified response triggered by dsRNA in the early embryo. Although dsRNA causes a global protein translation inhibition in a PKR eIF2a independent manner and leads to developmental delay and cell necrosis it also strongly induces p53 activation which then upregulates Interferon Stimulated Genes independently of interferon ligands. Importantly we demonstrate that the burst of p53 signaling dose not result in cell death but functions as a protective mechanism against deleterious translation blockage by slowing down global protein degradation via ISGylation. Our work has identified a distinct dsRNA induced stress response in the embryo reflecting an ancient innate immune memory before the establishment of the IFN system. It also raises the provocative question as to the original protective role of p53 during evolution. Overall design: To characterize the distribution of ribosomes on mRNA we performed Ribosome profiling Ribo seq analysis with RNA seq on dsRNA injected embryos.", null, null, null, "Zebrafish Riboseq high dose dsRNA2", "GSM8228804", null, "source name:whole embryo|tissue:whole embryo|developmental stage:50% epiboly stage|cell type:embryonic cell|genotype:wild type|treatment:dsRNA|geo loc name:missing|collection date:missing", "Zebrafish Riboseq high dose dsRNA2", "The library underwent quality control assessment and was subjected to Illumina Novaseq 6000 sequencing. Bio informatics analysis of RIBOseq profilling was performed to analyze the library data. Assembly: GRCz10 Supplementary files format and content: Excel file includes raw counts for each Sample Library strategy: Ribo seq", "whole embryo", null, "Both control group and dsRNA injected embryos at xxx hpf were thoroughly lysed on ice using lysis buffer.The lysate was then centrifuged at 12000g at 4\u00b0C for 10 minutes to remove cellular components including undigested material such as cell nuclei.Then RNaseI was added to the supernatant and incubated at 25\u00b0C for 30 minutes to digest RNA while the RNA fragments protected by ribosomes were preserved. Subsequently an RNA inhibitor was added to terminate the digestion reaction. The lysate was subjected to sucrose density gradient centrifugation and fractions containing single peaks at 260nm wavelength around the 80S monosome region were collected. The collected mixture was then thoroughly lysed using Trizol for RNA extraction. RNA fragments within the size range of 26 34nt was separated using polyacrylamide gel electrophoresis without xxx and the corresponding bands were collected. The recovered bands were subjected to ligation reaction where a preadenylylated and 3\u2019 blocked linker 5\u2019 rApp CTGTAGGCACCATCAAT NH2 3\u2019 was attached to the three prime end of the RNA. The ligated products were then recovered by performing polyacrylamide gel electrophoresis. Subsequently the recovered RNA fragments were reverse transcribed using a reverse primer to obtain extended cDNA molecules. The purified cDNA was subjected to circularization by using CircLigase Epicentre CL4111K resulting in the formation of circular cDNA molecules. To remove residual rRNA components in the cDNA a method involving hybridization with complementary primers specific to rRNA and subsequent heat denaturation was employed. The remaining circularized cDNA was then amplified through PCR and barcode sequences were incorporated.", null, "tissue:whole embryo|developmental stage:50% epiboly stage|cell type:embryonic cell|genotype:wild type|treatment:dsRNA", "GSM8228804", "GSM8228804: Zebrafish Riboseq high dose dsRNA2; Danio rerio; OTHER", "GSM8228804 r1", "GSM8228804", "1", "Both control group and dsRNA injected embryos at xxx hpf were thoroughly lysed on ice using lysis buffer.The lysate was then centrifuged at 12000g at 4\u00b0C for 10 minutes to remove cellular components including undigested material such as cell nuclei.Then RNaseI was added to the supernatant and incubated at 25\u00b0C for 30 minutes to digest RNA while the RNA fragments protected by ribosomes were preserved. Subsequently an RNA inhibitor was added to terminate the digestion reaction. The lysate was subjected to sucrose density gradient centrifugation and fractions containing single peaks at 260nm wavelength around the 80S monosome region were collected. The collected mixture was then thoroughly lysed using Trizol for RNA extraction. RNA fragments within the size range of 26 34nt was separated using polyacrylamide gel electrophoresis without xxx and the corresponding bands were collected. The recovered bands were subjected to ligation reaction where a preadenylylated and three prime blocked linker five prime rApp CTGTAGGCACCATCAAT NH2 three prime was attached to the three prime end of the RNA. The ligated products were then recovered by performing polyacrylamide gel electrophoresis. Subsequently the recovered RNA fragments were reverse transcribed using a reverse primer to obtain extended cDNA molecules. The purified cDNA was subjected to circularization by using CircLigase Epicentre CL4111K resulting in the formation of circular cDNA molecules. To remove residual rRNA components in the cDNA a method involving hybridization with complementary primers specific to rRNA and subsequent heat denaturation was employed. The remaining circularized cDNA was then amplified through PCR and barcode sequences were incorporated.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP503769", null, null, "dsR_2.R1.raw.fastq.gz dsR_2.R2.raw.fastq.gz", "fastq fastq", 15877367026.0, 52574063.0, "GSM8228804 r1", "0:151 1:151", "A:3017944891;C:2902425937;G:7224440180;T:2731881397;N:674621", 151, 151, null, null, 3017944891, 2902425937, 7224440180, 2731881397, 674621, "SRX24354554", "SRS21112322", "SRA1852128", "ShanDong University", "ShanDong University", null, null, null, null, null, null, null, null, null, null, null, "T", "T", "mates < 9% mapping rate", "illumina", "novaseq_era", "3prime", "random_priming", "unknown", "bulk", "unknown", "unknown", null, "China", "2024-04-24", "Gastrula", "Embryo", "Whole Organism", "All anatomical structures"], [31944, "SRR28790251", "SRX24354553", "SRS21112321", "SRP503769", "PRJNA1104180", "Double stranded RNA triggers a distinct integrated stress response in the early embryo [RNA seq Ribo seq]", "GSE265771", "Other", "Double stranded RNA dsRNA is associated with virus infections and is present as by products during the transcription of synthetic mRNA which has been widely used in gene gain of function studies and serves as a core component in emerging mRNA based therapies1 4. The presence of dsRNA in host cells induces an integrated stress response that functions to prevent virus replication and infection5 6. Unlike differentiated cells undifferentiated cells adopt a distinct defense strategy against RNA virus infection7 but the mechanism is unclear. We show a previously unidentified response triggered by dsRNA in the early embryo. Although dsRNA causes a global protein translation inhibition in a PKR eIF2a independent manner and leads to developmental delay and cell necrosis it also strongly induces p53 activation which then upregulates Interferon Stimulated Genes independently of interferon ligands. Importantly we demonstrate that the burst of p53 signaling dose not result in cell death but functions as a protective mechanism against deleterious translation blockage by slowing down global protein degradation via ISGylation. Our work has identified a distinct dsRNA induced stress response in the embryo reflecting an ancient innate immune memory before the establishment of the IFN system. It also raises the provocative question as to the original protective role of p53 during evolution. Overall design: To characterize the distribution of ribosomes on mRNA we performed Ribosome profiling Ribo seq analysis with RNA seq on dsRNA injected embryos.", null, null, null, "Zebrafish Riboseq high dose dsRNA1", "GSM8228803", null, "source name:whole embryo|tissue:whole embryo|developmental stage:50% epiboly stage|cell type:embryonic cell|genotype:wild type|treatment:dsRNA|geo loc name:missing|collection date:missing", "Zebrafish Riboseq high dose dsRNA1", "The library underwent quality control assessment and was subjected to Illumina Novaseq 6000 sequencing. Bio informatics analysis of RIBOseq profilling was performed to analyze the library data. Assembly: GRCz10 Supplementary files format and content: Excel file includes raw counts for each Sample Library strategy: Ribo seq", "whole embryo", null, "Both control group and dsRNA injected embryos at xxx hpf were thoroughly lysed on ice using lysis buffer.The lysate was then centrifuged at 12000g at 4\u00b0C for 10 minutes to remove cellular components including undigested material such as cell nuclei.Then RNaseI was added to the supernatant and incubated at 25\u00b0C for 30 minutes to digest RNA while the RNA fragments protected by ribosomes were preserved. Subsequently an RNA inhibitor was added to terminate the digestion reaction. The lysate was subjected to sucrose density gradient centrifugation and fractions containing single peaks at 260nm wavelength around the 80S monosome region were collected. The collected mixture was then thoroughly lysed using Trizol for RNA extraction. RNA fragments within the size range of 26 34nt was separated using polyacrylamide gel electrophoresis without xxx and the corresponding bands were collected. The recovered bands were subjected to ligation reaction where a preadenylylated and 3\u2019 blocked linker 5\u2019 rApp CTGTAGGCACCATCAAT NH2 3\u2019 was attached to the three prime end of the RNA. The ligated products were then recovered by performing polyacrylamide gel electrophoresis. Subsequently the recovered RNA fragments were reverse transcribed using a reverse primer to obtain extended cDNA molecules. The purified cDNA was subjected to circularization by using CircLigase Epicentre CL4111K resulting in the formation of circular cDNA molecules. To remove residual rRNA components in the cDNA a method involving hybridization with complementary primers specific to rRNA and subsequent heat denaturation was employed. The remaining circularized cDNA was then amplified through PCR and barcode sequences were incorporated.", null, "tissue:whole embryo|developmental stage:50% epiboly stage|cell type:embryonic cell|genotype:wild type|treatment:dsRNA", "GSM8228803", "GSM8228803: Zebrafish Riboseq high dose dsRNA1; Danio rerio; OTHER", "GSM8228803 r1", "GSM8228803", "1", "Both control group and dsRNA injected embryos at xxx hpf were thoroughly lysed on ice using lysis buffer.The lysate was then centrifuged at 12000g at 4\u00b0C for 10 minutes to remove cellular components including undigested material such as cell nuclei.Then RNaseI was added to the supernatant and incubated at 25\u00b0C for 30 minutes to digest RNA while the RNA fragments protected by ribosomes were preserved. Subsequently an RNA inhibitor was added to terminate the digestion reaction. The lysate was subjected to sucrose density gradient centrifugation and fractions containing single peaks at 260nm wavelength around the 80S monosome region were collected. The collected mixture was then thoroughly lysed using Trizol for RNA extraction. RNA fragments within the size range of 26 34nt was separated using polyacrylamide gel electrophoresis without xxx and the corresponding bands were collected. The recovered bands were subjected to ligation reaction where a preadenylylated and three prime blocked linker five prime rApp CTGTAGGCACCATCAAT NH2 three prime was attached to the three prime end of the RNA. The ligated products were then recovered by performing polyacrylamide gel electrophoresis. Subsequently the recovered RNA fragments were reverse transcribed using a reverse primer to obtain extended cDNA molecules. The purified cDNA was subjected to circularization by using CircLigase Epicentre CL4111K resulting in the formation of circular cDNA molecules. To remove residual rRNA components in the cDNA a method involving hybridization with complementary primers specific to rRNA and subsequent heat denaturation was employed. The remaining circularized cDNA was then amplified through PCR and barcode sequences were incorporated.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP503769", null, null, "dsR_1.R1.raw.fastq.gz dsR_1.R2.raw.fastq.gz", "fastq fastq", 13027928774.0, 43138837.0, "GSM8228803 r1", "0:151 1:151", "A:2538099201;C:2400262316;G:5852047273;T:2236961902;N:558082", 151, 151, null, null, 2538099201, 2400262316, 5852047273, 2236961902, 558082, "SRX24354553", "SRS21112321", "SRA1852128", "ShanDong University", "ShanDong University", null, null, null, null, null, null, null, null, null, null, null, "T", "T", "mates < 9% mapping rate", "illumina", "novaseq_era", "3prime", "random_priming", "unknown", "bulk", "unknown", "unknown", null, "China", "2024-04-24", "Gastrula", "Embryo", "Whole Organism", "All anatomical structures"], [31945, "SRR28790252", "SRX24354552", "SRS21112320", "SRP503769", "PRJNA1104180", "Double stranded RNA triggers a distinct integrated stress response in the early embryo [RNA seq Ribo seq]", "GSE265771", "Other", "Double stranded RNA dsRNA is associated with virus infections and is present as by products during the transcription of synthetic mRNA which has been widely used in gene gain of function studies and serves as a core component in emerging mRNA based therapies1 4. The presence of dsRNA in host cells induces an integrated stress response that functions to prevent virus replication and infection5 6. Unlike differentiated cells undifferentiated cells adopt a distinct defense strategy against RNA virus infection7 but the mechanism is unclear. We show a previously unidentified response triggered by dsRNA in the early embryo. Although dsRNA causes a global protein translation inhibition in a PKR eIF2a independent manner and leads to developmental delay and cell necrosis it also strongly induces p53 activation which then upregulates Interferon Stimulated Genes independently of interferon ligands. Importantly we demonstrate that the burst of p53 signaling dose not result in cell death but functions as a protective mechanism against deleterious translation blockage by slowing down global protein degradation via ISGylation. Our work has identified a distinct dsRNA induced stress response in the embryo reflecting an ancient innate immune memory before the establishment of the IFN system. It also raises the provocative question as to the original protective role of p53 during evolution. Overall design: To characterize the distribution of ribosomes on mRNA we performed Ribosome profiling Ribo seq analysis with RNA seq on dsRNA injected embryos.", null, null, null, "Zebrafish Riboseq high dose uninj2", "GSM8228802", null, "source name:whole embryo|tissue:whole embryo|developmental stage:50% epiboly stage|cell type:embryonic cell|genotype:wild type|treatment:uninjected|geo loc name:missing|collection date:missing", "Zebrafish Riboseq high dose uninj2", "The library underwent quality control assessment and was subjected to Illumina Novaseq 6000 sequencing. Bio informatics analysis of RIBOseq profilling was performed to analyze the library data. Assembly: GRCz10 Supplementary files format and content: Excel file includes raw counts for each Sample Library strategy: Ribo seq", "whole embryo", null, "Both control group and dsRNA injected embryos at xxx hpf were thoroughly lysed on ice using lysis buffer.The lysate was then centrifuged at 12000g at 4\u00b0C for 10 minutes to remove cellular components including undigested material such as cell nuclei.Then RNaseI was added to the supernatant and incubated at 25\u00b0C for 30 minutes to digest RNA while the RNA fragments protected by ribosomes were preserved. Subsequently an RNA inhibitor was added to terminate the digestion reaction. The lysate was subjected to sucrose density gradient centrifugation and fractions containing single peaks at 260nm wavelength around the 80S monosome region were collected. The collected mixture was then thoroughly lysed using Trizol for RNA extraction. RNA fragments within the size range of 26 34nt was separated using polyacrylamide gel electrophoresis without xxx and the corresponding bands were collected. The recovered bands were subjected to ligation reaction where a preadenylylated and 3\u2019 blocked linker 5\u2019 rApp CTGTAGGCACCATCAAT NH2 3\u2019 was attached to the three prime end of the RNA. The ligated products were then recovered by performing polyacrylamide gel electrophoresis. Subsequently the recovered RNA fragments were reverse transcribed using a reverse primer to obtain extended cDNA molecules. The purified cDNA was subjected to circularization by using CircLigase Epicentre CL4111K resulting in the formation of circular cDNA molecules. To remove residual rRNA components in the cDNA a method involving hybridization with complementary primers specific to rRNA and subsequent heat denaturation was employed. The remaining circularized cDNA was then amplified through PCR and barcode sequences were incorporated.", null, "tissue:whole embryo|developmental stage:50% epiboly stage|cell type:embryonic cell|genotype:wild type|treatment:uninjected", "GSM8228802", "GSM8228802: Zebrafish Riboseq high dose uninj2; Danio rerio; OTHER", "GSM8228802 r1", "GSM8228802", "1", "Both control group and dsRNA injected embryos at xxx hpf were thoroughly lysed on ice using lysis buffer.The lysate was then centrifuged at 12000g at 4\u00b0C for 10 minutes to remove cellular components including undigested material such as cell nuclei.Then RNaseI was added to the supernatant and incubated at 25\u00b0C for 30 minutes to digest RNA while the RNA fragments protected by ribosomes were preserved. Subsequently an RNA inhibitor was added to terminate the digestion reaction. The lysate was subjected to sucrose density gradient centrifugation and fractions containing single peaks at 260nm wavelength around the 80S monosome region were collected. The collected mixture was then thoroughly lysed using Trizol for RNA extraction. RNA fragments within the size range of 26 34nt was separated using polyacrylamide gel electrophoresis without xxx and the corresponding bands were collected. The recovered bands were subjected to ligation reaction where a preadenylylated and three prime blocked linker five prime rApp CTGTAGGCACCATCAAT NH2 three prime was attached to the three prime end of the RNA. The ligated products were then recovered by performing polyacrylamide gel electrophoresis. Subsequently the recovered RNA fragments were reverse transcribed using a reverse primer to obtain extended cDNA molecules. The purified cDNA was subjected to circularization by using CircLigase Epicentre CL4111K resulting in the formation of circular cDNA molecules. To remove residual rRNA components in the cDNA a method involving hybridization with complementary primers specific to rRNA and subsequent heat denaturation was employed. The remaining circularized cDNA was then amplified through PCR and barcode sequences were incorporated.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP503769", null, null, "un_2.R1.raw.fastq.gz un_2.R2.raw.fastq.gz", "fastq fastq", 13637060056.0, 45155828.0, "GSM8228802 r1", "0:151 1:151", "A:2596015831;C:2388366938;G:6201010197;T:2451085273;N:581817", 151, 151, null, null, 2596015831, 2388366938, 6201010197, 2451085273, 581817, "SRX24354552", "SRS21112320", "SRA1852128", "ShanDong University", "ShanDong University", null, null, null, null, null, null, null, null, null, null, null, "T", "T", "mates < 9% mapping rate", "illumina", "novaseq_era", "3prime", "random_priming", "unknown", "bulk", "unknown", "unknown", null, "China", "2024-04-24", "Gastrula", "Embryo", "Whole Organism", "All anatomical structures"], [31946, "SRR28790253", "SRX24354551", "SRS21112319", "SRP503769", "PRJNA1104180", "Double stranded RNA triggers a distinct integrated stress response in the early embryo [RNA seq Ribo seq]", "GSE265771", "Other", "Double stranded RNA dsRNA is associated with virus infections and is present as by products during the transcription of synthetic mRNA which has been widely used in gene gain of function studies and serves as a core component in emerging mRNA based therapies1 4. The presence of dsRNA in host cells induces an integrated stress response that functions to prevent virus replication and infection5 6. Unlike differentiated cells undifferentiated cells adopt a distinct defense strategy against RNA virus infection7 but the mechanism is unclear. We show a previously unidentified response triggered by dsRNA in the early embryo. Although dsRNA causes a global protein translation inhibition in a PKR eIF2a independent manner and leads to developmental delay and cell necrosis it also strongly induces p53 activation which then upregulates Interferon Stimulated Genes independently of interferon ligands. Importantly we demonstrate that the burst of p53 signaling dose not result in cell death but functions as a protective mechanism against deleterious translation blockage by slowing down global protein degradation via ISGylation. Our work has identified a distinct dsRNA induced stress response in the embryo reflecting an ancient innate immune memory before the establishment of the IFN system. It also raises the provocative question as to the original protective role of p53 during evolution. Overall design: To characterize the distribution of ribosomes on mRNA we performed Ribosome profiling Ribo seq analysis with RNA seq on dsRNA injected embryos.", null, null, null, "Zebrafish Riboseq high dose uninj1", "GSM8228801", null, "source name:whole embryo|tissue:whole embryo|developmental stage:50% epiboly stage|cell type:embryonic cell|genotype:wild type|treatment:uninjected|geo loc name:missing|collection date:missing", "Zebrafish Riboseq high dose uninj1", "The library underwent quality control assessment and was subjected to Illumina Novaseq 6000 sequencing. Bio informatics analysis of RIBOseq profilling was performed to analyze the library data. Assembly: GRCz10 Supplementary files format and content: Excel file includes raw counts for each Sample Library strategy: Ribo seq", "whole embryo", null, "Both control group and dsRNA injected embryos at xxx hpf were thoroughly lysed on ice using lysis buffer.The lysate was then centrifuged at 12000g at 4\u00b0C for 10 minutes to remove cellular components including undigested material such as cell nuclei.Then RNaseI was added to the supernatant and incubated at 25\u00b0C for 30 minutes to digest RNA while the RNA fragments protected by ribosomes were preserved. Subsequently an RNA inhibitor was added to terminate the digestion reaction. The lysate was subjected to sucrose density gradient centrifugation and fractions containing single peaks at 260nm wavelength around the 80S monosome region were collected. The collected mixture was then thoroughly lysed using Trizol for RNA extraction. RNA fragments within the size range of 26 34nt was separated using polyacrylamide gel electrophoresis without xxx and the corresponding bands were collected. The recovered bands were subjected to ligation reaction where a preadenylylated and 3\u2019 blocked linker 5\u2019 rApp CTGTAGGCACCATCAAT NH2 3\u2019 was attached to the three prime end of the RNA. The ligated products were then recovered by performing polyacrylamide gel electrophoresis. Subsequently the recovered RNA fragments were reverse transcribed using a reverse primer to obtain extended cDNA molecules. The purified cDNA was subjected to circularization by using CircLigase Epicentre CL4111K resulting in the formation of circular cDNA molecules. To remove residual rRNA components in the cDNA a method involving hybridization with complementary primers specific to rRNA and subsequent heat denaturation was employed. The remaining circularized cDNA was then amplified through PCR and barcode sequences were incorporated.", null, "tissue:whole embryo|developmental stage:50% epiboly stage|cell type:embryonic cell|genotype:wild type|treatment:uninjected", "GSM8228801", "GSM8228801: Zebrafish Riboseq high dose uninj1; Danio rerio; OTHER", "GSM8228801 r1", "GSM8228801", "1", "Both control group and dsRNA injected embryos at xxx hpf were thoroughly lysed on ice using lysis buffer.The lysate was then centrifuged at 12000g at 4\u00b0C for 10 minutes to remove cellular components including undigested material such as cell nuclei.Then RNaseI was added to the supernatant and incubated at 25\u00b0C for 30 minutes to digest RNA while the RNA fragments protected by ribosomes were preserved. Subsequently an RNA inhibitor was added to terminate the digestion reaction. The lysate was subjected to sucrose density gradient centrifugation and fractions containing single peaks at 260nm wavelength around the 80S monosome region were collected. The collected mixture was then thoroughly lysed using Trizol for RNA extraction. RNA fragments within the size range of 26 34nt was separated using polyacrylamide gel electrophoresis without xxx and the corresponding bands were collected. The recovered bands were subjected to ligation reaction where a preadenylylated and three prime blocked linker five prime rApp CTGTAGGCACCATCAAT NH2 three prime was attached to the three prime end of the RNA. The ligated products were then recovered by performing polyacrylamide gel electrophoresis. Subsequently the recovered RNA fragments were reverse transcribed using a reverse primer to obtain extended cDNA molecules. The purified cDNA was subjected to circularization by using CircLigase Epicentre CL4111K resulting in the formation of circular cDNA molecules. To remove residual rRNA components in the cDNA a method involving hybridization with complementary primers specific to rRNA and subsequent heat denaturation was employed. The remaining circularized cDNA was then amplified through PCR and barcode sequences were incorporated.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP503769", null, null, "un_1.R1.raw.fastq.gz un_1.R2.raw.fastq.gz", "fastq fastq", 13499333560.0, 44699780.0, "GSM8228801 r1", "0:151 1:151", "A:2587725963;C:2439789499;G:6080777505;T:2390460459;N:580134", 151, 151, null, null, 2587725963, 2439789499, 6080777505, 2390460459, 580134, "SRX24354551", "SRS21112319", "SRA1852128", "ShanDong University", "ShanDong University", null, null, null, null, null, null, null, null, null, null, null, "T", "T", "mates < 9% mapping rate", "illumina", "novaseq_era", "3prime", "random_priming", "unknown", "bulk", "unknown", "unknown", null, "China", "2024-04-24", "Gastrula", "Embryo", "Whole Organism", "All anatomical structures"], [31947, "SRR28790254", "SRX24354550", "SRS21112318", "SRP503769", "PRJNA1104180", "Double stranded RNA triggers a distinct integrated stress response in the early embryo [RNA seq Ribo seq]", "GSE265771", "Other", "Double stranded RNA dsRNA is associated with virus infections and is present as by products during the transcription of synthetic mRNA which has been widely used in gene gain of function studies and serves as a core component in emerging mRNA based therapies1 4. The presence of dsRNA in host cells induces an integrated stress response that functions to prevent virus replication and infection5 6. Unlike differentiated cells undifferentiated cells adopt a distinct defense strategy against RNA virus infection7 but the mechanism is unclear. We show a previously unidentified response triggered by dsRNA in the early embryo. Although dsRNA causes a global protein translation inhibition in a PKR eIF2a independent manner and leads to developmental delay and cell necrosis it also strongly induces p53 activation which then upregulates Interferon Stimulated Genes independently of interferon ligands. Importantly we demonstrate that the burst of p53 signaling dose not result in cell death but functions as a protective mechanism against deleterious translation blockage by slowing down global protein degradation via ISGylation. Our work has identified a distinct dsRNA induced stress response in the embryo reflecting an ancient innate immune memory before the establishment of the IFN system. It also raises the provocative question as to the original protective role of p53 during evolution. Overall design: To characterize the distribution of ribosomes on mRNA we performed Ribosome profiling Ribo seq analysis with RNA seq on dsRNA injected embryos.", null, null, null, "Zebrafish Riboseq low dose dsRNA2", "GSM8228800", null, "source name:whole embryo|tissue:whole embryo|developmental stage:50% epiboly stage|cell type:embryonic cell|genotype:wild type|treatment:dsRNA|geo loc name:missing|collection date:missing", "Zebrafish Riboseq low dose dsRNA2", "The library underwent quality control assessment and was subjected to Illumina Novaseq 6000 sequencing. Bio informatics analysis of RIBOseq profilling was performed to analyze the library data. Assembly: GRCz10 Supplementary files format and content: Excel file includes raw counts for each Sample Library strategy: Ribo seq", "whole embryo", null, "Both control group and dsRNA injected embryos at xxx hpf were thoroughly lysed on ice using lysis buffer.The lysate was then centrifuged at 12000g at 4\u00b0C for 10 minutes to remove cellular components including undigested material such as cell nuclei.Then RNaseI was added to the supernatant and incubated at 25\u00b0C for 30 minutes to digest RNA while the RNA fragments protected by ribosomes were preserved. Subsequently an RNA inhibitor was added to terminate the digestion reaction. The lysate was subjected to sucrose density gradient centrifugation and fractions containing single peaks at 260nm wavelength around the 80S monosome region were collected. The collected mixture was then thoroughly lysed using Trizol for RNA extraction. RNA fragments within the size range of 26 34nt was separated using polyacrylamide gel electrophoresis without xxx and the corresponding bands were collected. The recovered bands were subjected to ligation reaction where a preadenylylated and 3\u2019 blocked linker 5\u2019 rApp CTGTAGGCACCATCAAT NH2 3\u2019 was attached to the three prime end of the RNA. The ligated products were then recovered by performing polyacrylamide gel electrophoresis. Subsequently the recovered RNA fragments were reverse transcribed using a reverse primer to obtain extended cDNA molecules. The purified cDNA was subjected to circularization by using CircLigase Epicentre CL4111K resulting in the formation of circular cDNA molecules. To remove residual rRNA components in the cDNA a method involving hybridization with complementary primers specific to rRNA and subsequent heat denaturation was employed. The remaining circularized cDNA was then amplified through PCR and barcode sequences were incorporated.", null, "tissue:whole embryo|developmental stage:50% epiboly stage|cell type:embryonic cell|genotype:wild type|treatment:dsRNA", "GSM8228800", "GSM8228800: Zebrafish Riboseq low dose dsRNA2; Danio rerio; OTHER", "GSM8228800 r1", "GSM8228800", "1", "Both control group and dsRNA injected embryos at xxx hpf were thoroughly lysed on ice using lysis buffer.The lysate was then centrifuged at 12000g at 4\u00b0C for 10 minutes to remove cellular components including undigested material such as cell nuclei.Then RNaseI was added to the supernatant and incubated at 25\u00b0C for 30 minutes to digest RNA while the RNA fragments protected by ribosomes were preserved. Subsequently an RNA inhibitor was added to terminate the digestion reaction. The lysate was subjected to sucrose density gradient centrifugation and fractions containing single peaks at 260nm wavelength around the 80S monosome region were collected. The collected mixture was then thoroughly lysed using Trizol for RNA extraction. RNA fragments within the size range of 26 34nt was separated using polyacrylamide gel electrophoresis without xxx and the corresponding bands were collected. The recovered bands were subjected to ligation reaction where a preadenylylated and three prime blocked linker five prime rApp CTGTAGGCACCATCAAT NH2 three prime was attached to the three prime end of the RNA. The ligated products were then recovered by performing polyacrylamide gel electrophoresis. Subsequently the recovered RNA fragments were reverse transcribed using a reverse primer to obtain extended cDNA molecules. The purified cDNA was subjected to circularization by using CircLigase Epicentre CL4111K resulting in the formation of circular cDNA molecules. To remove residual rRNA components in the cDNA a method involving hybridization with complementary primers specific to rRNA and subsequent heat denaturation was employed. The remaining circularized cDNA was then amplified through PCR and barcode sequences were incorporated.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP503769", null, null, "dsR_2.raw.1.fastq.gz dsR_2.raw.2.fastq.gz", "fastq fastq", 13335544766.0, 44157433.0, "GSM8228800 r1", "0:151 1:151", "A:2383528724;C:2262795235;G:6593680813;T:2094730217;N:809777", 151, 151, null, null, 2383528724, 2262795235, 6593680813, 2094730217, 809777, "SRX24354550", "SRS21112318", "SRA1852128", "ShanDong University", "ShanDong University", null, null, null, null, null, null, null, null, null, null, null, "T", "T", "mates < 9% mapping rate", "illumina", "novaseq_era", "3prime", "random_priming", "unknown", "bulk", "unknown", "unknown", null, "China", "2024-04-24", "Gastrula", "Embryo", "Whole Organism", "All anatomical structures"], [31948, "SRR28790255", "SRX24354549", "SRS21112317", "SRP503769", "PRJNA1104180", "Double stranded RNA triggers a distinct integrated stress response in the early embryo [RNA seq Ribo seq]", "GSE265771", "Other", "Double stranded RNA dsRNA is associated with virus infections and is present as by products during the transcription of synthetic mRNA which has been widely used in gene gain of function studies and serves as a core component in emerging mRNA based therapies1 4. The presence of dsRNA in host cells induces an integrated stress response that functions to prevent virus replication and infection5 6. Unlike differentiated cells undifferentiated cells adopt a distinct defense strategy against RNA virus infection7 but the mechanism is unclear. We show a previously unidentified response triggered by dsRNA in the early embryo. Although dsRNA causes a global protein translation inhibition in a PKR eIF2a independent manner and leads to developmental delay and cell necrosis it also strongly induces p53 activation which then upregulates Interferon Stimulated Genes independently of interferon ligands. Importantly we demonstrate that the burst of p53 signaling dose not result in cell death but functions as a protective mechanism against deleterious translation blockage by slowing down global protein degradation via ISGylation. Our work has identified a distinct dsRNA induced stress response in the embryo reflecting an ancient innate immune memory before the establishment of the IFN system. It also raises the provocative question as to the original protective role of p53 during evolution. Overall design: To characterize the distribution of ribosomes on mRNA we performed Ribosome profiling Ribo seq analysis with RNA seq on dsRNA injected embryos.", null, null, null, "Zebrafish Riboseq low dose dsRNA1", "GSM8228799", null, "source name:whole embryo|tissue:whole embryo|developmental stage:50% epiboly stage|cell type:embryonic cell|genotype:wild type|treatment:dsRNA|geo loc name:missing|collection date:missing", "Zebrafish Riboseq low dose dsRNA1", "The library underwent quality control assessment and was subjected to Illumina Novaseq 6000 sequencing. Bio informatics analysis of RIBOseq profilling was performed to analyze the library data. Assembly: GRCz10 Supplementary files format and content: Excel file includes raw counts for each Sample Library strategy: Ribo seq", "whole embryo", null, "Both control group and dsRNA injected embryos at xxx hpf were thoroughly lysed on ice using lysis buffer.The lysate was then centrifuged at 12000g at 4\u00b0C for 10 minutes to remove cellular components including undigested material such as cell nuclei.Then RNaseI was added to the supernatant and incubated at 25\u00b0C for 30 minutes to digest RNA while the RNA fragments protected by ribosomes were preserved. Subsequently an RNA inhibitor was added to terminate the digestion reaction. The lysate was subjected to sucrose density gradient centrifugation and fractions containing single peaks at 260nm wavelength around the 80S monosome region were collected. The collected mixture was then thoroughly lysed using Trizol for RNA extraction. RNA fragments within the size range of 26 34nt was separated using polyacrylamide gel electrophoresis without xxx and the corresponding bands were collected. The recovered bands were subjected to ligation reaction where a preadenylylated and 3\u2019 blocked linker 5\u2019 rApp CTGTAGGCACCATCAAT NH2 3\u2019 was attached to the three prime end of the RNA. The ligated products were then recovered by performing polyacrylamide gel electrophoresis. Subsequently the recovered RNA fragments were reverse transcribed using a reverse primer to obtain extended cDNA molecules. The purified cDNA was subjected to circularization by using CircLigase Epicentre CL4111K resulting in the formation of circular cDNA molecules. To remove residual rRNA components in the cDNA a method involving hybridization with complementary primers specific to rRNA and subsequent heat denaturation was employed. The remaining circularized cDNA was then amplified through PCR and barcode sequences were incorporated.", null, "tissue:whole embryo|developmental stage:50% epiboly stage|cell type:embryonic cell|genotype:wild type|treatment:dsRNA", "GSM8228799", "GSM8228799: Zebrafish Riboseq low dose dsRNA1; Danio rerio; OTHER", "GSM8228799 r1", "GSM8228799", "1", "Both control group and dsRNA injected embryos at xxx hpf were thoroughly lysed on ice using lysis buffer.The lysate was then centrifuged at 12000g at 4\u00b0C for 10 minutes to remove cellular components including undigested material such as cell nuclei.Then RNaseI was added to the supernatant and incubated at 25\u00b0C for 30 minutes to digest RNA while the RNA fragments protected by ribosomes were preserved. Subsequently an RNA inhibitor was added to terminate the digestion reaction. The lysate was subjected to sucrose density gradient centrifugation and fractions containing single peaks at 260nm wavelength around the 80S monosome region were collected. The collected mixture was then thoroughly lysed using Trizol for RNA extraction. RNA fragments within the size range of 26 34nt was separated using polyacrylamide gel electrophoresis without xxx and the corresponding bands were collected. The recovered bands were subjected to ligation reaction where a preadenylylated and three prime blocked linker five prime rApp CTGTAGGCACCATCAAT NH2 three prime was attached to the three prime end of the RNA. The ligated products were then recovered by performing polyacrylamide gel electrophoresis. Subsequently the recovered RNA fragments were reverse transcribed using a reverse primer to obtain extended cDNA molecules. The purified cDNA was subjected to circularization by using CircLigase Epicentre CL4111K resulting in the formation of circular cDNA molecules. To remove residual rRNA components in the cDNA a method involving hybridization with complementary primers specific to rRNA and subsequent heat denaturation was employed. The remaining circularized cDNA was then amplified through PCR and barcode sequences were incorporated.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP503769", null, null, "dsR_1.raw.1.fastq.gz dsR_1.raw.2.fastq.gz", "fastq fastq", 12530433302.0, 41491501.0, "GSM8228799 r1", "0:151 1:151", "A:2300207705;C:2164355626;G:6050556625;T:2014558360;N:754986", 151, 151, null, null, 2300207705, 2164355626, 6050556625, 2014558360, 754986, "SRX24354549", "SRS21112317", "SRA1852128", "ShanDong University", "ShanDong University", null, null, null, null, null, null, null, null, null, null, null, "T", "T", "mates < 9% mapping rate", "illumina", "novaseq_era", "3prime", "random_priming", "unknown", "bulk", "unknown", "unknown", null, "China", "2024-04-24", "Gastrula", "Embryo", "Whole Organism", "All anatomical structures"], [31949, "SRR28790256", "SRX24354548", "SRS21112316", "SRP503769", "PRJNA1104180", "Double stranded RNA triggers a distinct integrated stress response in the early embryo [RNA seq Ribo seq]", "GSE265771", "Other", "Double stranded RNA dsRNA is associated with virus infections and is present as by products during the transcription of synthetic mRNA which has been widely used in gene gain of function studies and serves as a core component in emerging mRNA based therapies1 4. The presence of dsRNA in host cells induces an integrated stress response that functions to prevent virus replication and infection5 6. Unlike differentiated cells undifferentiated cells adopt a distinct defense strategy against RNA virus infection7 but the mechanism is unclear. We show a previously unidentified response triggered by dsRNA in the early embryo. Although dsRNA causes a global protein translation inhibition in a PKR eIF2a independent manner and leads to developmental delay and cell necrosis it also strongly induces p53 activation which then upregulates Interferon Stimulated Genes independently of interferon ligands. Importantly we demonstrate that the burst of p53 signaling dose not result in cell death but functions as a protective mechanism against deleterious translation blockage by slowing down global protein degradation via ISGylation. Our work has identified a distinct dsRNA induced stress response in the embryo reflecting an ancient innate immune memory before the establishment of the IFN system. It also raises the provocative question as to the original protective role of p53 during evolution. Overall design: To characterize the distribution of ribosomes on mRNA we performed Ribosome profiling Ribo seq analysis with RNA seq on dsRNA injected embryos.", null, null, null, "Zebrafish Riboseq low dose uninj2", "GSM8228798", null, "source name:whole embryo|tissue:whole embryo|developmental stage:50% epiboly stage|cell type:embryonic cell|genotype:wild type|treatment:uninjected|geo loc name:missing|collection date:missing", "Zebrafish Riboseq low dose uninj2", "The library underwent quality control assessment and was subjected to Illumina Novaseq 6000 sequencing. Bio informatics analysis of RIBOseq profilling was performed to analyze the library data. Assembly: GRCz10 Supplementary files format and content: Excel file includes raw counts for each Sample Library strategy: Ribo seq", "whole embryo", null, "Both control group and dsRNA injected embryos at xxx hpf were thoroughly lysed on ice using lysis buffer.The lysate was then centrifuged at 12000g at 4\u00b0C for 10 minutes to remove cellular components including undigested material such as cell nuclei.Then RNaseI was added to the supernatant and incubated at 25\u00b0C for 30 minutes to digest RNA while the RNA fragments protected by ribosomes were preserved. Subsequently an RNA inhibitor was added to terminate the digestion reaction. The lysate was subjected to sucrose density gradient centrifugation and fractions containing single peaks at 260nm wavelength around the 80S monosome region were collected. The collected mixture was then thoroughly lysed using Trizol for RNA extraction. RNA fragments within the size range of 26 34nt was separated using polyacrylamide gel electrophoresis without xxx and the corresponding bands were collected. The recovered bands were subjected to ligation reaction where a preadenylylated and 3\u2019 blocked linker 5\u2019 rApp CTGTAGGCACCATCAAT NH2 3\u2019 was attached to the three prime end of the RNA. The ligated products were then recovered by performing polyacrylamide gel electrophoresis. Subsequently the recovered RNA fragments were reverse transcribed using a reverse primer to obtain extended cDNA molecules. The purified cDNA was subjected to circularization by using CircLigase Epicentre CL4111K resulting in the formation of circular cDNA molecules. To remove residual rRNA components in the cDNA a method involving hybridization with complementary primers specific to rRNA and subsequent heat denaturation was employed. The remaining circularized cDNA was then amplified through PCR and barcode sequences were incorporated.", null, "tissue:whole embryo|developmental stage:50% epiboly stage|cell type:embryonic cell|genotype:wild type|treatment:uninjected", "GSM8228798", "GSM8228798: Zebrafish Riboseq low dose uninj2; Danio rerio; OTHER", "GSM8228798 r1", "GSM8228798", "1", "Both control group and dsRNA injected embryos at xxx hpf were thoroughly lysed on ice using lysis buffer.The lysate was then centrifuged at 12000g at 4\u00b0C for 10 minutes to remove cellular components including undigested material such as cell nuclei.Then RNaseI was added to the supernatant and incubated at 25\u00b0C for 30 minutes to digest RNA while the RNA fragments protected by ribosomes were preserved. Subsequently an RNA inhibitor was added to terminate the digestion reaction. The lysate was subjected to sucrose density gradient centrifugation and fractions containing single peaks at 260nm wavelength around the 80S monosome region were collected. The collected mixture was then thoroughly lysed using Trizol for RNA extraction. RNA fragments within the size range of 26 34nt was separated using polyacrylamide gel electrophoresis without xxx and the corresponding bands were collected. The recovered bands were subjected to ligation reaction where a preadenylylated and three prime blocked linker five prime rApp CTGTAGGCACCATCAAT NH2 three prime was attached to the three prime end of the RNA. The ligated products were then recovered by performing polyacrylamide gel electrophoresis. Subsequently the recovered RNA fragments were reverse transcribed using a reverse primer to obtain extended cDNA molecules. The purified cDNA was subjected to circularization by using CircLigase Epicentre CL4111K resulting in the formation of circular cDNA molecules. To remove residual rRNA components in the cDNA a method involving hybridization with complementary primers specific to rRNA and subsequent heat denaturation was employed. The remaining circularized cDNA was then amplified through PCR and barcode sequences were incorporated.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP503769", null, null, "un_2.raw.1.fastq.gz un_2.raw.2.fastq.gz", "fastq fastq", 12132018896.0, 40172248.0, "GSM8228798 r1", "0:151 1:151", "A:2225485331;C:2027579165;G:5953072417;T:1925141784;N:740199", 151, 151, null, null, 2225485331, 2027579165, 5953072417, 1925141784, 740199, "SRX24354548", "SRS21112316", "SRA1852128", "ShanDong University", "ShanDong University", null, null, null, null, null, null, null, null, null, null, null, "T", "T", "mates < 9% mapping rate", "illumina", "novaseq_era", "3prime", "random_priming", "unknown", "bulk", "unknown", "unknown", null, "China", "2024-04-24", "Gastrula", "Embryo", "Whole Organism", "All anatomical structures"], [31950, "SRR28790257", "SRX24354547", "SRS21112315", "SRP503769", "PRJNA1104180", "Double stranded RNA triggers a distinct integrated stress response in the early embryo [RNA seq Ribo seq]", "GSE265771", "Other", "Double stranded RNA dsRNA is associated with virus infections and is present as by products during the transcription of synthetic mRNA which has been widely used in gene gain of function studies and serves as a core component in emerging mRNA based therapies1 4. The presence of dsRNA in host cells induces an integrated stress response that functions to prevent virus replication and infection5 6. Unlike differentiated cells undifferentiated cells adopt a distinct defense strategy against RNA virus infection7 but the mechanism is unclear. We show a previously unidentified response triggered by dsRNA in the early embryo. Although dsRNA causes a global protein translation inhibition in a PKR eIF2a independent manner and leads to developmental delay and cell necrosis it also strongly induces p53 activation which then upregulates Interferon Stimulated Genes independently of interferon ligands. Importantly we demonstrate that the burst of p53 signaling dose not result in cell death but functions as a protective mechanism against deleterious translation blockage by slowing down global protein degradation via ISGylation. Our work has identified a distinct dsRNA induced stress response in the embryo reflecting an ancient innate immune memory before the establishment of the IFN system. It also raises the provocative question as to the original protective role of p53 during evolution. Overall design: To characterize the distribution of ribosomes on mRNA we performed Ribosome profiling Ribo seq analysis with RNA seq on dsRNA injected embryos.", null, null, null, "Zebrafish Riboseq low dose uninj1", "GSM8228797", null, "source name:whole embryo|tissue:whole embryo|developmental stage:50% epiboly stage|cell type:embryonic cell|genotype:wild type|treatment:uninjected|geo loc name:missing|collection date:missing", "Zebrafish Riboseq low dose uninj1", "The library underwent quality control assessment and was subjected to Illumina Novaseq 6000 sequencing. Bio informatics analysis of RIBOseq profilling was performed to analyze the library data. Assembly: GRCz10 Supplementary files format and content: Excel file includes raw counts for each Sample Library strategy: Ribo seq", "whole embryo", null, "Both control group and dsRNA injected embryos at xxx hpf were thoroughly lysed on ice using lysis buffer.The lysate was then centrifuged at 12000g at 4\u00b0C for 10 minutes to remove cellular components including undigested material such as cell nuclei.Then RNaseI was added to the supernatant and incubated at 25\u00b0C for 30 minutes to digest RNA while the RNA fragments protected by ribosomes were preserved. Subsequently an RNA inhibitor was added to terminate the digestion reaction. The lysate was subjected to sucrose density gradient centrifugation and fractions containing single peaks at 260nm wavelength around the 80S monosome region were collected. The collected mixture was then thoroughly lysed using Trizol for RNA extraction. RNA fragments within the size range of 26 34nt was separated using polyacrylamide gel electrophoresis without xxx and the corresponding bands were collected. The recovered bands were subjected to ligation reaction where a preadenylylated and 3\u2019 blocked linker 5\u2019 rApp CTGTAGGCACCATCAAT NH2 3\u2019 was attached to the three prime end of the RNA. The ligated products were then recovered by performing polyacrylamide gel electrophoresis. Subsequently the recovered RNA fragments were reverse transcribed using a reverse primer to obtain extended cDNA molecules. The purified cDNA was subjected to circularization by using CircLigase Epicentre CL4111K resulting in the formation of circular cDNA molecules. To remove residual rRNA components in the cDNA a method involving hybridization with complementary primers specific to rRNA and subsequent heat denaturation was employed. The remaining circularized cDNA was then amplified through PCR and barcode sequences were incorporated.", null, "tissue:whole embryo|developmental stage:50% epiboly stage|cell type:embryonic cell|genotype:wild type|treatment:uninjected", "GSM8228797", "GSM8228797: Zebrafish Riboseq low dose uninj1; Danio rerio; OTHER", "GSM8228797 r1", "GSM8228797", "1", "Both control group and dsRNA injected embryos at xxx hpf were thoroughly lysed on ice using lysis buffer.The lysate was then centrifuged at 12000g at 4\u00b0C for 10 minutes to remove cellular components including undigested material such as cell nuclei.Then RNaseI was added to the supernatant and incubated at 25\u00b0C for 30 minutes to digest RNA while the RNA fragments protected by ribosomes were preserved. Subsequently an RNA inhibitor was added to terminate the digestion reaction. The lysate was subjected to sucrose density gradient centrifugation and fractions containing single peaks at 260nm wavelength around the 80S monosome region were collected. The collected mixture was then thoroughly lysed using Trizol for RNA extraction. RNA fragments within the size range of 26 34nt was separated using polyacrylamide gel electrophoresis without xxx and the corresponding bands were collected. The recovered bands were subjected to ligation reaction where a preadenylylated and three prime blocked linker five prime rApp CTGTAGGCACCATCAAT NH2 three prime was attached to the three prime end of the RNA. The ligated products were then recovered by performing polyacrylamide gel electrophoresis. Subsequently the recovered RNA fragments were reverse transcribed using a reverse primer to obtain extended cDNA molecules. The purified cDNA was subjected to circularization by using CircLigase Epicentre CL4111K resulting in the formation of circular cDNA molecules. To remove residual rRNA components in the cDNA a method involving hybridization with complementary primers specific to rRNA and subsequent heat denaturation was employed. The remaining circularized cDNA was then amplified through PCR and barcode sequences were incorporated.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP503769", null, null, "un_1.raw.1.fastq.gz un_1.raw.2.fastq.gz", "fastq fastq", 10423454802.0, 34514751.0, "GSM8228797 r1", "0:151 1:151", "A:1931094519;C:1788894999;G:5028533789;T:1674294221;N:637274", 151, 151, null, null, 1931094519, 1788894999, 5028533789, 1674294221, 637274, "SRX24354547", "SRS21112315", "SRA1852128", "ShanDong University", "ShanDong University", null, null, null, null, null, null, null, null, null, null, null, "T", "T", "mates < 9% mapping rate", "illumina", "novaseq_era", "3prime", "random_priming", "unknown", "bulk", "unknown", "unknown", null, "China", "2024-04-24", "Gastrula", "Embryo", "Whole Organism", "All anatomical structures"], [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"], [35567, "SRR32928199", "SRX28203119", "SRS24552825", "SRP575522", "PRJNA1244443", "An improved high yield method for isolating nuclei from individual zebrafish embryos for single nucleus RNA sequencing", "PRJNA1244443", "Other", "Zebrafish are an ideal system to study the effects of chemical genetic and environmental perturbations on development due to their high fecundity and fast growth. Recently single cell sequencing has emerged as a powerful tool to measure the effect of these perturbations at a whole embryo scale. These types of experiments rely on the ability to isolate nuclei from a large number of individually barcoded zebrafish embryos in parallel. Here we report a method for efficiently isolating high quality nuclei from zebrafish embryos in a 96 well plate format by bead homogenization in a lysis buffer. Through head to head sciPlex RNA seq experiments we demonstrate that this method represents a substantial improvement over enzymatic dissociation and that it is compatible with a wide range of developmental stages.", null, null, "Sci plexRNAseq of a 48hpf whole zebrafish embryo dissociated using enzymatic digestion. This subpool was sequenced on a large number of cells in order to determine the cell number and diversity captured by the method.", null, "expt2.broad.48.enzymatic.P01.F01.fq.gz", null, "seq depth:broad many cells|Experiment id:2|dissociation method:enzymatic|replicate:6|strain:AB|isolate:not applicable|breed:not applicable|cultivar:not applicable|ecotype:not applicable|age:48 hpf|dev stage:48 hpf|collection date:2024 01 31|geo loc name:USA:Washington Seattle|sex:not collected|tissue:Whole Embryo|sample type:Whole organism|BioSampleModel:Model organism or animal", null, null, null, null, null, null, null, null, "sciPlex RNAseq of whole Danio rerio embryos: expt2 broad sequencing of many cells at low depth 48hpf enzymatic dissociation replicate 6", "100", "100", "Libraries were generated using sciPlex RNAseq3 combinatorial indexing. Each fasta represents a sample split from a large multiplexed sequencing run. Cell IDs are in the header of each fastq.", null, null, "OTHER", "TRANSCRIPTOMIC SINGLE CELL", "other", "SINGLE", "ILLUMINA", "NextSeq 2000", null, "SRP575522", null, null, "expt2.broad.48.enzymatic.P01.F01.fq.gz", "fastq", 800436.0, 9529.0, "expt2.broad.48.enzymatic.P01.F01.fq.gz", "0:84", "A:247920;C:168170;G:180799;T:203153;N:394", 84, null, null, null, 247920, 168170, 180799, 203153, 394, "SRX28203119", "SRS24552825", "SRA2104467", "University of Washington|Genome Sciences", "University of Washington", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq_v2", "unknown", "other", "unknown", "sc", "single_cell_generic", "generic-scrnaseq-only", null, "United States", "2025-03-31", "Hatching", "Embryo", "Whole Organism", "All anatomical structures"], [35568, "SRR32928200", "SRX28203118", "SRS24552823", "SRP575522", "PRJNA1244443", "An improved high yield method for isolating nuclei from individual zebrafish embryos for single nucleus RNA sequencing", "PRJNA1244443", "Other", "Zebrafish are an ideal system to study the effects of chemical genetic and environmental perturbations on development due to their high fecundity and fast growth. Recently single cell sequencing has emerged as a powerful tool to measure the effect of these perturbations at a whole embryo scale. These types of experiments rely on the ability to isolate nuclei from a large number of individually barcoded zebrafish embryos in parallel. Here we report a method for efficiently isolating high quality nuclei from zebrafish embryos in a 96 well plate format by bead homogenization in a lysis buffer. Through head to head sciPlex RNA seq experiments we demonstrate that this method represents a substantial improvement over enzymatic dissociation and that it is compatible with a wide range of developmental stages.", null, null, "Sci plexRNAseq of a 48hpf whole zebrafish embryo dissociated using enzymatic digestion. This subpool was sequenced on a large number of cells in order to determine the cell number and diversity captured by the method.", null, "expt2.broad.48.enzymatic.P01.E01.fq.gz", null, "seq depth:broad many cells|Experiment id:2|dissociation method:enzymatic|replicate:5|strain:AB|isolate:not applicable|breed:not applicable|cultivar:not applicable|ecotype:not applicable|age:48 hpf|dev stage:48 hpf|collection date:2024 01 31|geo loc name:USA:Washington Seattle|sex:not collected|tissue:Whole Embryo|sample type:Whole organism|BioSampleModel:Model organism or animal", null, null, null, null, null, null, null, null, "sciPlex RNAseq of whole Danio rerio embryos: expt2 broad sequencing of many cells at low depth 48hpf enzymatic dissociation replicate 5", "99", "99", "Libraries were generated using sciPlex RNAseq3 combinatorial indexing. Each fasta represents a sample split from a large multiplexed sequencing run. Cell IDs are in the header of each fastq.", null, null, "OTHER", "TRANSCRIPTOMIC SINGLE CELL", "other", "SINGLE", "ILLUMINA", "NextSeq 2000", null, "SRP575522", null, null, "expt2.broad.48.enzymatic.P01.E01.fq.gz", "fastq", 496356.0, 5909.0, "expt2.broad.48.enzymatic.P01.E01.fq.gz", "0:84", "A:156600;C:99183;G:113282;T:127036;N:255", 84, null, null, null, 156600, 99183, 113282, 127036, 255, "SRX28203118", "SRS24552823", "SRA2104467", "University of Washington|Genome Sciences", "University of Washington", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq_v2", "unknown", "other", "unknown", "sc", "single_cell_generic", "generic-scrnaseq-only", null, "United States", "2025-03-31", "Hatching", "Embryo", "Whole Organism", "All anatomical structures"], [35569, "SRR32928201", "SRX28203117", "SRS24552824", "SRP575522", "PRJNA1244443", "An improved high yield method for isolating nuclei from individual zebrafish embryos for single nucleus RNA sequencing", "PRJNA1244443", "Other", "Zebrafish are an ideal system to study the effects of chemical genetic and environmental perturbations on development due to their high fecundity and fast growth. Recently single cell sequencing has emerged as a powerful tool to measure the effect of these perturbations at a whole embryo scale. These types of experiments rely on the ability to isolate nuclei from a large number of individually barcoded zebrafish embryos in parallel. Here we report a method for efficiently isolating high quality nuclei from zebrafish embryos in a 96 well plate format by bead homogenization in a lysis buffer. Through head to head sciPlex RNA seq experiments we demonstrate that this method represents a substantial improvement over enzymatic dissociation and that it is compatible with a wide range of developmental stages.", null, null, "Sci plexRNAseq of a 48hpf whole zebrafish embryo dissociated using enzymatic digestion. This subpool was sequenced on a large number of cells in order to determine the cell number and diversity captured by the method.", null, "expt2.broad.48.enzymatic.P01.D01.fq.gz", null, "seq depth:broad many cells|Experiment id:2|dissociation method:enzymatic|replicate:4|strain:AB|isolate:not applicable|breed:not applicable|cultivar:not applicable|ecotype:not applicable|age:48 hpf|dev stage:48 hpf|collection date:2024 01 31|geo loc name:USA:Washington Seattle|sex:not collected|tissue:Whole Embryo|sample type:Whole organism|BioSampleModel:Model organism or animal", null, null, null, null, null, null, null, null, "sciPlex RNAseq of whole Danio rerio embryos: expt2 broad sequencing of many cells at low depth 48hpf enzymatic dissociation replicate 4", "98", "98", "Libraries were generated using sciPlex RNAseq3 combinatorial indexing. Each fasta represents a sample split from a large multiplexed sequencing run. 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Through head to head sciPlex RNA seq experiments we demonstrate that this method represents a substantial improvement over enzymatic dissociation and that it is compatible with a wide range of developmental stages.", null, null, "Sci plexRNAseq of a 96hpf whole zebrafish embryo dissociated using bead homogenization.", null, "expt3.96.bead.P18.H11.fq.gz", null, "seq depth:NA|Experiment id:3|dissociation method:bead|replicate:8|strain:AB|isolate:not applicable|breed:not applicable|cultivar:not applicable|ecotype:not applicable|age:96 hpf|dev stage:96 hpf|collection date:2024 03 19|geo loc name:USA:Washington Seattle|sex:not collected|tissue:Whole Embryo|sample type:Whole organism|BioSampleModel:Model organism or animal", null, null, null, null, null, null, null, null, "sciPlex RNAseq of whole Danio rerio embryos: expt3 96hpf bead dissociation replicate 8", "205", "205", "Libraries were generated using sciPlex RNAseq3 combinatorial indexing. 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Through head to head sciPlex RNA seq experiments we demonstrate that this method represents a substantial improvement over enzymatic dissociation and that it is compatible with a wide range of developmental stages.", null, null, "Sci plexRNAseq of a 96hpf whole zebrafish embryo dissociated using bead homogenization.", null, "expt3.96.bead.P18.G11.fq.gz", null, "seq depth:NA|Experiment id:3|dissociation method:bead|replicate:7|strain:AB|isolate:not applicable|breed:not applicable|cultivar:not applicable|ecotype:not applicable|age:96 hpf|dev stage:96 hpf|collection date:2024 03 19|geo loc name:USA:Washington Seattle|sex:not collected|tissue:Whole Embryo|sample type:Whole organism|BioSampleModel:Model organism or animal", null, null, null, null, null, null, null, null, "sciPlex RNAseq of whole Danio rerio embryos: expt3 96hpf bead dissociation replicate 7", "204", "204", "Libraries were generated using sciPlex RNAseq3 combinatorial indexing. 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