metadata: run_metadata: 187 rows where devstage_curation = "Multi-stage" and experiment.library

187 rows where devstage_curation = "Multi-stage" and experiment.library_selection = "other"

descending

Link	rowid ▼	run.accession	experiment.accession	sample.accession	study.accession	bioproject	study.title	study.alias	study.type	study.abstract	study.attributes	study.PMIDs	sample.description	sample.title	sample.alias	sample.centername	sample.attributes	GEOsample.title	GEOsample.dataprocessing	GEOsample.source	GEOsample.treatmentprotocol	GEOsample.extractprotocol	GEOsample.growthprotocol	GEOsample.characteristics	GEOsample.accession	experiment.title	experiment.alias	experiment.library_name	experiment.design_description	experiment.library_construction_protocol	experiment.library_strategy	experiment.library_source	experiment.library_selection	experiment.library_layout	experiment.platform	experiment.instrument_model	experiment.study_ref	run.title	run.attributes	run.filename	run.semantic_name	run.total_bases	run.total_spots	run.alias	run.read_lengths	run.base_counts	run.r1_length	run.r2_length	run.r3_length	run.r4_length	run.Acount	run.Ccount	run.Gcount	run.Tcount	run.Ncount	run.experiment	run.pool_member	submission.accession	submission.srasource	submission.bioprojectsource	seqdetective.n_mates	seqdetective.mapping_rate.mate1	seqdetective.mapping_rate.mate2	seqdetective.nofeature_rate.mate1	seqdetective.nofeature_rate.mate2	seqdetective.sparsity.mate1	seqdetective.sparsity.mate2	seqdetective.pos_strand_rate.mate1	seqdetective.pos_strand_rate.mate2	seqdetective.readlen.mate1	seqdetective.readlen.mate2	seqdetective.judgement.mate1	seqdetective.judgement.mate2	seqdetective.judgement.reason	platform_family	instrument_generation	read_bias	selection_class	prep_kit	sc_or_bulk	tech_class	technology	submission.bioprojectsource.country	earliest_date	devstage_curation	devstage_curation_coarse	tissue_curation	tissue_curation_coarse
11042	11042	ERR9839781	ERX9385638	ERS12199238	ERP138294	PRJEB53494	Nano3P seq: transcriptome wide analysis of gene expression and tail dynamics using end capture nanopore cDNA sequencing	94bf5509-4622-4d5f-b7c5-6a14bdfac340	Other	RNA polyadenylation plays a central role in RNA maturation fate and stability. In response to developmental cues polyA tail lengths can vary affecting the translation efficiency and stability of mRNAs. Here we develop Nanopore three prime end capture sequencing Nano3P seq a novel method that relies on nanopore cDNA sequencing to simultaneously quantify RNA abundance tail composition and tail length dynamics at per read resolution. By employing a template switching based sequencing protocol Nano3P seq can sequence any given RNA molecule from its three prime end regardless of its polyadenylation status without xxx need for PCR amplification or ligation of RNA adapters. We demonstrate that Nano3P seq captures a wide diversity of RNA biotypes providing quantitative estimates of RNA abundance and tail lengths in mRNA lncRNA sn/snoRNA scaRNA and rRNA molecules. We find that in addition to mRNA and lncRNA polyA tails can be identified in 16S mitochondrial rRNA in both mouse and zebrafish models. Moreover we show that mRNA tail lengths are dynamically regulated during vertebrate embryogenesis at an isoform specific level correlating with mRNA decay. Finally we identify non A bases within polyA tails of various lengths and reveal their distribution during vertebrate embryogenesis. Overall Nano3P seq is a simple and robust method for accurately estimating transcript levels tail lengths and tail composition heterogeneity in individual reads with minimal library preparation biases both in the coding and non coding transcriptome.	ENA FIRST PUBLIC:2022 10 10\|ENA LAST UPDATE:2022 10 10		Zebrafish Ribodepleted RNA 2hpf 4hpf 6hpf	Zebrafish Ribodepleted Rep3	SAMEA110100413	CENTER FOR GENOMIC REGULATION (CRG)	ENA FIRST PUBLIC:2022 10 10\|ENA LAST UPDATE:2022 10 10\|External Id:SAMEA110100413\|INSDC center alias:CENTER FOR GENOMIC REGULATION CRG\|INSDC center name:CENTER FOR GENOMIC REGULATION CRG\|INSDC first public:2022 10 10T00:20:53Z\|INSDC last update:2022 10 10T00:20:53Z\|INSDC status:public\|Submitter Id:Zebrafish Ribodepleted Rep3\|common name:zebrafish\|sample name:Zebrafish Ribodepleted Rep3									MinION sequencing	ena EXPERIMENT TAB 13 06 2022 16:07:52:807 817	cDNA897892 ZFRDR3	1		RNA-Seq	TRANSCRIPTOMIC	other	SINGLE	OXFORD_NANOPORE	MinION	ERP138294	MinION sequencing	ENA FIRST PUBLIC:2022 10 10\|ENA LAST UPDATE:2022 10 10	cDNA897892_ZFRDR3.tar.gz	nanopore	848659575.0	587586.0	ena RUN TAB 13 06 2022 16:07:52:808 818	0:1444.32	A:210205014;C:186527780;G:188214279;T:263712502;N:0	1444				210205014	186527780	188214279	263712502	0	ERX9385638	ERS12199238	ERA15547404	CENTER FOR GENOMIC REGULATION (CRG)\|European Nucleotide Archive	CENTER FOR GENOMIC REGULATION (CRG)															ont	ont	3prime	poly_a	unknown	bulk	unknown	unknown	Spain	2022-10-10	Multi-stage	Embryo	Undetermined	Embryo Imprecise
11043	11043	ERR9839780	ERX9385637	ERS12199237	ERP138294	PRJEB53494	Nano3P seq: transcriptome wide analysis of gene expression and tail dynamics using end capture nanopore cDNA sequencing	94bf5509-4622-4d5f-b7c5-6a14bdfac340	Other	RNA polyadenylation plays a central role in RNA maturation fate and stability. In response to developmental cues polyA tail lengths can vary affecting the translation efficiency and stability of mRNAs. Here we develop Nanopore three prime end capture sequencing Nano3P seq a novel method that relies on nanopore cDNA sequencing to simultaneously quantify RNA abundance tail composition and tail length dynamics at per read resolution. By employing a template switching based sequencing protocol Nano3P seq can sequence any given RNA molecule from its three prime end regardless of its polyadenylation status without xxx need for PCR amplification or ligation of RNA adapters. We demonstrate that Nano3P seq captures a wide diversity of RNA biotypes providing quantitative estimates of RNA abundance and tail lengths in mRNA lncRNA sn/snoRNA scaRNA and rRNA molecules. We find that in addition to mRNA and lncRNA polyA tails can be identified in 16S mitochondrial rRNA in both mouse and zebrafish models. Moreover we show that mRNA tail lengths are dynamically regulated during vertebrate embryogenesis at an isoform specific level correlating with mRNA decay. Finally we identify non A bases within polyA tails of various lengths and reveal their distribution during vertebrate embryogenesis. Overall Nano3P seq is a simple and robust method for accurately estimating transcript levels tail lengths and tail composition heterogeneity in individual reads with minimal library preparation biases both in the coding and non coding transcriptome.	ENA FIRST PUBLIC:2022 10 10\|ENA LAST UPDATE:2022 10 10		Zebrafish Ribodepleted RNA 2hpf 4hpf 6hpf	Zebrafish Ribodepleted Rep2	SAMEA110100412	CENTER FOR GENOMIC REGULATION (CRG)	ENA FIRST PUBLIC:2022 10 10\|ENA LAST UPDATE:2022 10 10\|External Id:SAMEA110100412\|INSDC center alias:CENTER FOR GENOMIC REGULATION CRG\|INSDC center name:CENTER FOR GENOMIC REGULATION CRG\|INSDC first public:2022 10 10T00:20:53Z\|INSDC last update:2022 10 10T00:20:53Z\|INSDC status:public\|Submitter Id:Zebrafish Ribodepleted Rep2\|common name:zebrafish\|sample name:Zebrafish Ribodepleted Rep2									MinION sequencing	ena EXPERIMENT TAB 13 06 2022 16:07:52:807 815	cDNA123791 ZFRDR2	1		RNA-Seq	TRANSCRIPTOMIC	other	SINGLE	OXFORD_NANOPORE	MinION	ERP138294	MinION sequencing	ENA FIRST PUBLIC:2022 10 10\|ENA LAST UPDATE:2022 10 10	cDNA123791_ZFRDR2.tar.gz	nanopore	2229275175.0	1955617.0	ena RUN TAB 13 06 2022 16:07:52:807 816	0:1139.93	A:533543922;C:498938137;G:515833119;T:680959997;N:0	1139				533543922	498938137	515833119	680959997	0	ERX9385637	ERS12199237	ERA15547404	CENTER FOR GENOMIC REGULATION (CRG)\|European Nucleotide Archive	CENTER FOR GENOMIC REGULATION (CRG)															ont	ont	3prime	poly_a	unknown	bulk	unknown	unknown	Spain	2022-10-10	Multi-stage	Embryo	Undetermined	Embryo Imprecise
11044	11044	ERR9839779	ERX9385636	ERS12199236	ERP138294	PRJEB53494	Nano3P seq: transcriptome wide analysis of gene expression and tail dynamics using end capture nanopore cDNA sequencing	94bf5509-4622-4d5f-b7c5-6a14bdfac340	Other	RNA polyadenylation plays a central role in RNA maturation fate and stability. In response to developmental cues polyA tail lengths can vary affecting the translation efficiency and stability of mRNAs. Here we develop Nanopore three prime end capture sequencing Nano3P seq a novel method that relies on nanopore cDNA sequencing to simultaneously quantify RNA abundance tail composition and tail length dynamics at per read resolution. By employing a template switching based sequencing protocol Nano3P seq can sequence any given RNA molecule from its three prime end regardless of its polyadenylation status without xxx need for PCR amplification or ligation of RNA adapters. We demonstrate that Nano3P seq captures a wide diversity of RNA biotypes providing quantitative estimates of RNA abundance and tail lengths in mRNA lncRNA sn/snoRNA scaRNA and rRNA molecules. We find that in addition to mRNA and lncRNA polyA tails can be identified in 16S mitochondrial rRNA in both mouse and zebrafish models. Moreover we show that mRNA tail lengths are dynamically regulated during vertebrate embryogenesis at an isoform specific level correlating with mRNA decay. Finally we identify non A bases within polyA tails of various lengths and reveal their distribution during vertebrate embryogenesis. Overall Nano3P seq is a simple and robust method for accurately estimating transcript levels tail lengths and tail composition heterogeneity in individual reads with minimal library preparation biases both in the coding and non coding transcriptome.	ENA FIRST PUBLIC:2022 10 10\|ENA LAST UPDATE:2022 10 10		Zebrafish Ribodepleted RNA 2hpf 4hpf 6hpf	Zebrafish Ribodepleted Rep1	SAMEA110100411	CENTER FOR GENOMIC REGULATION (CRG)	ENA FIRST PUBLIC:2022 10 10\|ENA LAST UPDATE:2022 10 10\|External Id:SAMEA110100411\|INSDC center alias:CENTER FOR GENOMIC REGULATION CRG\|INSDC center name:CENTER FOR GENOMIC REGULATION CRG\|INSDC first public:2022 10 10T00:20:53Z\|INSDC last update:2022 10 10T00:20:53Z\|INSDC status:public\|Submitter Id:Zebrafish Ribodepleted Rep1\|common name:zebrafish\|sample name:Zebrafish Ribodepleted Rep1									MinION sequencing	ena EXPERIMENT TAB 13 06 2022 16:07:52:807 813	cDNA786327 ZFRDR1	1		RNA-Seq	TRANSCRIPTOMIC	other	SINGLE	OXFORD_NANOPORE	MinION	ERP138294	MinION sequencing	ENA FIRST PUBLIC:2022 10 10\|ENA LAST UPDATE:2022 10 10	cDNA786327_ZFRDR1.tar.gz	nanopore	1900613556.0	1660167.0	ena RUN TAB 13 06 2022 16:07:52:807 814	0:1144.83	A:473050820;C:438054520;G:425169743;T:564338473;N:0	1144				473050820	438054520	425169743	564338473	0	ERX9385636	ERS12199236	ERA15547404	CENTER FOR GENOMIC REGULATION (CRG)\|European Nucleotide Archive	CENTER FOR GENOMIC REGULATION (CRG)															ont	ont	3prime	poly_a	unknown	bulk	unknown	unknown	Spain	2022-10-10	Multi-stage	Embryo	Undetermined	Embryo Imprecise
25182	25182	SRR25670729	SRX21396042	SRS18636200	SRP455680	PRJNA1006406	Control of polyA tail length and translation in vertebrate oocytes and early embryos	GSE241107	Other	During oocyte maturation and early embryonic development polyA tail lengths strongly influence mRNA translation. However how tail lengths are controlled at different developmental stages has been unclear. Here we performed tail length and translational profiling of mRNA reporter libraries each with > 10 million three prime UTR sequence variants in frog oocytes and embryos and fish embryos. These analyses revealed that the UUUUA motif specifies cytoplasmic polyadenylation and identified diverse context features that modulate the activity of this 5 mer. Additional sequence motifs drive stage specific deadenylation in embryos and UUUUA and C rich motifs drive tail length independent translational repression in oocytes. A neural network model accurately predicts tail length change during oocyte maturation in frogs mice and humans. Analyses of human sequence variants showed that those predicted to disrupt tail length control have been under negative selection implying that our insights into control of polyA tail length and translation have implications for human health and fertility. Overall design: Sythetic mRNA reporter libraries with random three prime UTR sequences under four different sequence contexts were injected into frog oocytes and embryos and fish embryos. PolyA tail lengths were measured to at different developmental stages to examine sequence motifs that caused tail length changes. At the same time polyA tail lengths of endogenous mRNAs from frog oocytes and embryos fish embryos and mouse oocytes were measured to investiage how their tail lengths were controlled. Please note that sample titles have been updated on Jan 6 2024.				Fish embryo mRNA germ ring PAL seq v4	GSM7716871		source name:embryo\|tissue:embryo\|treatment:N1\|geo loc name:missing\|collection date:missing	Fish embryo mRNA germ ring PAL seq v4	For gene specific tail seq of mRNA reporters data were processed with a custom script available at https://github.com/coffeebond/MPRA tail seq. For PAL seq v3 or v4 reads were trimmed with cutadapt v3.7 with the parameters “ m 15 quality base=64 q 20 20 match read wildcards e 0.05 a NNNNATCTCGTATGCCGTCTTCTGCTTG O 7”. The trimmed reads were mapped using STAR v2.7.1a to the reference database containing the genomic sequences of the organism from which the mRNAs were obtained the genomic sequences of humans or fish depending on which spike in RNAs were used and the sequences of the polyA standards generated previously with the parameters “ runThreadN 16 runMode alignReads outFilterMultimapNmax 1 outReadsUnmapped Fastx outFilterType BySJout outSAMattributes All outSAMtype BAM Unsorted SortedByCoordinate”. Uniquely mapped read were furhter processed with a custom script available at https://github.com/coffeebond/PAL seq. Assembly: Homo sapiens: GRCh38.p7 primary assembly. Mus muculus: GRCh38.p4 primary assembly. Xenopus laevis: v10.1 assembly. Danio rerio: GRCz11 assembly. Supplementary files format and content: For gene specific tail seq of mRNA reporters tab delimited text files with columns indicating 1 sequence of the variable region; 2 median tail length; 3 number of reads; 4–7 tail lengths at quantile 10 25 75 and 90. Supplementary files format and content: For PAL seq v3 or v4 tab delimited files with columns indicating 1 gene ID or polyA site ID; 2 read cluster ID; 3 tail length Library strategy: PAL seq v4	embryo		Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5 100 mM KCl 5 mM MgCl2 1% [v/v] Triton X 100 100 µg/ml cycloheximide cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer] and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr denuded with 3 mg/ml hyaluronidase for 2 min washed and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases but for the N60 library injected into fish embryos and frog oocytes the N37 PAS N17 library injected into fish embryos and frog oocytes and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009 8 pmol KXSH010 and 2x SSC 0.3 M NaCl 30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher 65002 and incubated for 20 min at 23°C on a thermal mixer shaking with 15 sec on and 1 min 45 sec off. The superna…		tissue:embryo\|treatment:N1	GSM7716871	GSM7716871: Fish embryo mRNA germ ring PAL seq v4; Danio rerio; OTHER	GSM7716871 r1	GSM7716871	1	Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5 100 mM KCl 5 mM MgCl2 1% [v/v] Triton X 100 100 µg/ml cycloheximide cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer] and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr denuded with 3 mg/ml hyaluronidase for 2 min washed and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases but for the N60 library injected into fish embryos and frog oocytes the N37 PAS N17 library injected into fish embryos and frog oocytes and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009 8 pmol KXSH010 and 2x SSC 0.3 M NaCl 30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher 65002 and incubated for 20 min at 23°C on a thermal mixer shaking with 15 sec on and 1 min 45 sec off. The superna…	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	Illumina HiSeq 2500	SRP455680			Fish_embryo_mRNA_germ_ring_PAL_seq_v4_rep1_raw_read2.fastq.gz Fish_embryo_mRNA_germ_ring_PAL_seq_v4_rep1_raw_read1.fastq.gz	fastq fastq	2834842912.0	9234016.0	GSM7716871 r1	0:52 1:255	A:725811118;C:702410592;G:747583409;T:651018348;N:8019445	52	255			725811118	702410592	747583409	651018348	8019445	SRX21396042	SRS18636200	SRA1694849	Whitehead Institute	Whitehead Institute	2	0.00023	0.30494	8e-05	0.01793	0.99967	0.99971	0.5	1.0	52	255	T	B	mate1 technical by mapping diff	illumina	hiseq_era	unknown	poly_a	unknown	bulk	bulk	bulk	United States	2023-08-17	Multi-stage	Embryo	Embryo Imprecise	All anatomical structures
25183	25183	SRR25670730	SRX21396042	SRS18636200	SRP455680	PRJNA1006406	Control of polyA tail length and translation in vertebrate oocytes and early embryos	GSE241107	Other	During oocyte maturation and early embryonic development polyA tail lengths strongly influence mRNA translation. However how tail lengths are controlled at different developmental stages has been unclear. Here we performed tail length and translational profiling of mRNA reporter libraries each with > 10 million three prime UTR sequence variants in frog oocytes and embryos and fish embryos. These analyses revealed that the UUUUA motif specifies cytoplasmic polyadenylation and identified diverse context features that modulate the activity of this 5 mer. Additional sequence motifs drive stage specific deadenylation in embryos and UUUUA and C rich motifs drive tail length independent translational repression in oocytes. A neural network model accurately predicts tail length change during oocyte maturation in frogs mice and humans. Analyses of human sequence variants showed that those predicted to disrupt tail length control have been under negative selection implying that our insights into control of polyA tail length and translation have implications for human health and fertility. Overall design: Sythetic mRNA reporter libraries with random three prime UTR sequences under four different sequence contexts were injected into frog oocytes and embryos and fish embryos. PolyA tail lengths were measured to at different developmental stages to examine sequence motifs that caused tail length changes. At the same time polyA tail lengths of endogenous mRNAs from frog oocytes and embryos fish embryos and mouse oocytes were measured to investiage how their tail lengths were controlled. Please note that sample titles have been updated on Jan 6 2024.				Fish embryo mRNA germ ring PAL seq v4	GSM7716871		source name:embryo\|tissue:embryo\|treatment:N1\|geo loc name:missing\|collection date:missing	Fish embryo mRNA germ ring PAL seq v4	For gene specific tail seq of mRNA reporters data were processed with a custom script available at https://github.com/coffeebond/MPRA tail seq. For PAL seq v3 or v4 reads were trimmed with cutadapt v3.7 with the parameters “ m 15 quality base=64 q 20 20 match read wildcards e 0.05 a NNNNATCTCGTATGCCGTCTTCTGCTTG O 7”. The trimmed reads were mapped using STAR v2.7.1a to the reference database containing the genomic sequences of the organism from which the mRNAs were obtained the genomic sequences of humans or fish depending on which spike in RNAs were used and the sequences of the polyA standards generated previously with the parameters “ runThreadN 16 runMode alignReads outFilterMultimapNmax 1 outReadsUnmapped Fastx outFilterType BySJout outSAMattributes All outSAMtype BAM Unsorted SortedByCoordinate”. Uniquely mapped read were furhter processed with a custom script available at https://github.com/coffeebond/PAL seq. Assembly: Homo sapiens: GRCh38.p7 primary assembly. Mus muculus: GRCh38.p4 primary assembly. Xenopus laevis: v10.1 assembly. Danio rerio: GRCz11 assembly. Supplementary files format and content: For gene specific tail seq of mRNA reporters tab delimited text files with columns indicating 1 sequence of the variable region; 2 median tail length; 3 number of reads; 4–7 tail lengths at quantile 10 25 75 and 90. Supplementary files format and content: For PAL seq v3 or v4 tab delimited files with columns indicating 1 gene ID or polyA site ID; 2 read cluster ID; 3 tail length Library strategy: PAL seq v4	embryo		Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5 100 mM KCl 5 mM MgCl2 1% [v/v] Triton X 100 100 µg/ml cycloheximide cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer] and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr denuded with 3 mg/ml hyaluronidase for 2 min washed and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases but for the N60 library injected into fish embryos and frog oocytes the N37 PAS N17 library injected into fish embryos and frog oocytes and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009 8 pmol KXSH010 and 2x SSC 0.3 M NaCl 30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher 65002 and incubated for 20 min at 23°C on a thermal mixer shaking with 15 sec on and 1 min 45 sec off. The superna…		tissue:embryo\|treatment:N1	GSM7716871	GSM7716871: Fish embryo mRNA germ ring PAL seq v4; Danio rerio; OTHER	GSM7716871 r1	GSM7716871	1	Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5 100 mM KCl 5 mM MgCl2 1% [v/v] Triton X 100 100 µg/ml cycloheximide cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer] and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr denuded with 3 mg/ml hyaluronidase for 2 min washed and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases but for the N60 library injected into fish embryos and frog oocytes the N37 PAS N17 library injected into fish embryos and frog oocytes and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009 8 pmol KXSH010 and 2x SSC 0.3 M NaCl 30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher 65002 and incubated for 20 min at 23°C on a thermal mixer shaking with 15 sec on and 1 min 45 sec off. The superna…	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	Illumina HiSeq 2500	SRP455680			Fish_embryo_mRNA_germ_ring_PAL_seq_v4_rep2_raw_read1.fastq.gz Fish_embryo_mRNA_germ_ring_PAL_seq_v4_rep2_raw_read2.fastq.gz	fastq fastq	3473033898.0	11312814.0	GSM7716871 r2	0:52 1:255	A:853178471;C:899976159;G:962100712;T:750811461;N:6967095	52	255			853178471	899976159	962100712	750811461	6967095	SRX21396042	SRS18636200	SRA1694849	Whitehead Institute	Whitehead Institute	2	0.00049	0.0	0.00012	0.0	0.99941	1.0	0.64864		52	255	T	T	mates < 9% mapping rate	illumina	hiseq_era	unknown	poly_a	unknown	bulk	bulk	bulk	United States	2023-08-17	Multi-stage	Embryo	Embryo Imprecise	All anatomical structures
25184	25184	SRR25670731	SRX21396041	SRS18636199	SRP455680	PRJNA1006406	Control of polyA tail length and translation in vertebrate oocytes and early embryos	GSE241107	Other	During oocyte maturation and early embryonic development polyA tail lengths strongly influence mRNA translation. However how tail lengths are controlled at different developmental stages has been unclear. Here we performed tail length and translational profiling of mRNA reporter libraries each with > 10 million three prime UTR sequence variants in frog oocytes and embryos and fish embryos. These analyses revealed that the UUUUA motif specifies cytoplasmic polyadenylation and identified diverse context features that modulate the activity of this 5 mer. Additional sequence motifs drive stage specific deadenylation in embryos and UUUUA and C rich motifs drive tail length independent translational repression in oocytes. A neural network model accurately predicts tail length change during oocyte maturation in frogs mice and humans. Analyses of human sequence variants showed that those predicted to disrupt tail length control have been under negative selection implying that our insights into control of polyA tail length and translation have implications for human health and fertility. Overall design: Sythetic mRNA reporter libraries with random three prime UTR sequences under four different sequence contexts were injected into frog oocytes and embryos and fish embryos. PolyA tail lengths were measured to at different developmental stages to examine sequence motifs that caused tail length changes. At the same time polyA tail lengths of endogenous mRNAs from frog oocytes and embryos fish embryos and mouse oocytes were measured to investiage how their tail lengths were controlled. Please note that sample titles have been updated on Jan 6 2024.				Fish embryo mRNA zfs:0000015 PAL seq v4	GSM7716870		source name:embryo\|tissue:embryo\|treatment:N1\|geo loc name:missing\|collection date:missing	Fish embryo mRNA zfs:0000015 PAL seq v4	For gene specific tail seq of mRNA reporters data were processed with a custom script available at https://github.com/coffeebond/MPRA tail seq. For PAL seq v3 or v4 reads were trimmed with cutadapt v3.7 with the parameters “ m 15 quality base=64 q 20 20 match read wildcards e 0.05 a NNNNATCTCGTATGCCGTCTTCTGCTTG O 7”. The trimmed reads were mapped using STAR v2.7.1a to the reference database containing the genomic sequences of the organism from which the mRNAs were obtained the genomic sequences of humans or fish depending on which spike in RNAs were used and the sequences of the polyA standards generated previously with the parameters “ runThreadN 16 runMode alignReads outFilterMultimapNmax 1 outReadsUnmapped Fastx outFilterType BySJout outSAMattributes All outSAMtype BAM Unsorted SortedByCoordinate”. Uniquely mapped read were furhter processed with a custom script available at https://github.com/coffeebond/PAL seq. Assembly: Homo sapiens: GRCh38.p7 primary assembly. Mus muculus: GRCh38.p4 primary assembly. Xenopus laevis: v10.1 assembly. Danio rerio: GRCz11 assembly. Supplementary files format and content: For gene specific tail seq of mRNA reporters tab delimited text files with columns indicating 1 sequence of the variable region; 2 median tail length; 3 number of reads; 4–7 tail lengths at quantile 10 25 75 and 90. Supplementary files format and content: For PAL seq v3 or v4 tab delimited files with columns indicating 1 gene ID or polyA site ID; 2 read cluster ID; 3 tail length Library strategy: PAL seq v4	embryo		Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5 100 mM KCl 5 mM MgCl2 1% [v/v] Triton X 100 100 µg/ml cycloheximide cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer] and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr denuded with 3 mg/ml hyaluronidase for 2 min washed and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases but for the N60 library injected into fish embryos and frog oocytes the N37 PAS N17 library injected into fish embryos and frog oocytes and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009 8 pmol KXSH010 and 2x SSC 0.3 M NaCl 30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher 65002 and incubated for 20 min at 23°C on a thermal mixer shaking with 15 sec on and 1 min 45 sec off. The superna…		tissue:embryo\|treatment:N1	GSM7716870	GSM7716870: Fish embryo mRNA zfs:0000015 PAL seq v4; Danio rerio; OTHER	GSM7716870 r1	GSM7716870	1	Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5 100 mM KCl 5 mM MgCl2 1% [v/v] Triton X 100 100 µg/ml cycloheximide cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer] and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr denuded with 3 mg/ml hyaluronidase for 2 min washed and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases but for the N60 library injected into fish embryos and frog oocytes the N37 PAS N17 library injected into fish embryos and frog oocytes and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009 8 pmol KXSH010 and 2x SSC 0.3 M NaCl 30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher 65002 and incubated for 20 min at 23°C on a thermal mixer shaking with 15 sec on and 1 min 45 sec off. The superna…	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	Illumina HiSeq 2500	SRP455680			Fish_embryo_mRNA_30percent_epiboly_PAL_seq_v4_rep1_raw_read1.fastq.gz Fish_embryo_mRNA_30percent_epiboly_PAL_seq_v4_rep1_raw_read2.fastq.gz	fastq fastq	2695203962.0	8779166.0	GSM7716870 r1	0:52 1:255	A:684535386;C:669852151;G:719008886;T:614209811;N:7597728	52	255			684535386	669852151	719008886	614209811	7597728	SRX21396041	SRS18636199	SRA1694849	Whitehead Institute	Whitehead Institute	2	0.00117	0.35837	0.00017	0.00682	0.99859	0.99963	0.69473	1.0	52	255	T	B	mate1 technical by mapping diff	illumina	hiseq_era	unknown	poly_a	unknown	bulk	bulk	bulk	United States	2023-08-17	Multi-stage	Embryo	Embryo Imprecise	All anatomical structures
25185	25185	SRR25670732	SRX21396041	SRS18636199	SRP455680	PRJNA1006406	Control of polyA tail length and translation in vertebrate oocytes and early embryos	GSE241107	Other	During oocyte maturation and early embryonic development polyA tail lengths strongly influence mRNA translation. However how tail lengths are controlled at different developmental stages has been unclear. Here we performed tail length and translational profiling of mRNA reporter libraries each with > 10 million three prime UTR sequence variants in frog oocytes and embryos and fish embryos. These analyses revealed that the UUUUA motif specifies cytoplasmic polyadenylation and identified diverse context features that modulate the activity of this 5 mer. Additional sequence motifs drive stage specific deadenylation in embryos and UUUUA and C rich motifs drive tail length independent translational repression in oocytes. A neural network model accurately predicts tail length change during oocyte maturation in frogs mice and humans. Analyses of human sequence variants showed that those predicted to disrupt tail length control have been under negative selection implying that our insights into control of polyA tail length and translation have implications for human health and fertility. Overall design: Sythetic mRNA reporter libraries with random three prime UTR sequences under four different sequence contexts were injected into frog oocytes and embryos and fish embryos. PolyA tail lengths were measured to at different developmental stages to examine sequence motifs that caused tail length changes. At the same time polyA tail lengths of endogenous mRNAs from frog oocytes and embryos fish embryos and mouse oocytes were measured to investiage how their tail lengths were controlled. Please note that sample titles have been updated on Jan 6 2024.				Fish embryo mRNA zfs:0000015 PAL seq v4	GSM7716870		source name:embryo\|tissue:embryo\|treatment:N1\|geo loc name:missing\|collection date:missing	Fish embryo mRNA zfs:0000015 PAL seq v4	For gene specific tail seq of mRNA reporters data were processed with a custom script available at https://github.com/coffeebond/MPRA tail seq. For PAL seq v3 or v4 reads were trimmed with cutadapt v3.7 with the parameters “ m 15 quality base=64 q 20 20 match read wildcards e 0.05 a NNNNATCTCGTATGCCGTCTTCTGCTTG O 7”. The trimmed reads were mapped using STAR v2.7.1a to the reference database containing the genomic sequences of the organism from which the mRNAs were obtained the genomic sequences of humans or fish depending on which spike in RNAs were used and the sequences of the polyA standards generated previously with the parameters “ runThreadN 16 runMode alignReads outFilterMultimapNmax 1 outReadsUnmapped Fastx outFilterType BySJout outSAMattributes All outSAMtype BAM Unsorted SortedByCoordinate”. Uniquely mapped read were furhter processed with a custom script available at https://github.com/coffeebond/PAL seq. Assembly: Homo sapiens: GRCh38.p7 primary assembly. Mus muculus: GRCh38.p4 primary assembly. Xenopus laevis: v10.1 assembly. Danio rerio: GRCz11 assembly. Supplementary files format and content: For gene specific tail seq of mRNA reporters tab delimited text files with columns indicating 1 sequence of the variable region; 2 median tail length; 3 number of reads; 4–7 tail lengths at quantile 10 25 75 and 90. Supplementary files format and content: For PAL seq v3 or v4 tab delimited files with columns indicating 1 gene ID or polyA site ID; 2 read cluster ID; 3 tail length Library strategy: PAL seq v4	embryo		Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5 100 mM KCl 5 mM MgCl2 1% [v/v] Triton X 100 100 µg/ml cycloheximide cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer] and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr denuded with 3 mg/ml hyaluronidase for 2 min washed and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases but for the N60 library injected into fish embryos and frog oocytes the N37 PAS N17 library injected into fish embryos and frog oocytes and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009 8 pmol KXSH010 and 2x SSC 0.3 M NaCl 30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher 65002 and incubated for 20 min at 23°C on a thermal mixer shaking with 15 sec on and 1 min 45 sec off. The superna…		tissue:embryo\|treatment:N1	GSM7716870	GSM7716870: Fish embryo mRNA zfs:0000015 PAL seq v4; Danio rerio; OTHER	GSM7716870 r1	GSM7716870	1	Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5 100 mM KCl 5 mM MgCl2 1% [v/v] Triton X 100 100 µg/ml cycloheximide cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer] and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr denuded with 3 mg/ml hyaluronidase for 2 min washed and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases but for the N60 library injected into fish embryos and frog oocytes the N37 PAS N17 library injected into fish embryos and frog oocytes and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009 8 pmol KXSH010 and 2x SSC 0.3 M NaCl 30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher 65002 and incubated for 20 min at 23°C on a thermal mixer shaking with 15 sec on and 1 min 45 sec off. The superna…	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	Illumina HiSeq 2500	SRP455680			Fish_embryo_mRNA_30percent_epiboly_PAL_seq_v4_rep2_raw_read2.fastq.gz Fish_embryo_mRNA_30percent_epiboly_PAL_seq_v4_rep2_raw_read1.fastq.gz	fastq fastq	3476338446.0	11323578.0	GSM7716870 r2	0:52 1:255	A:841832234;C:898325199;G:968774318;T:760398070;N:7008625	52	255			841832234	898325199	968774318	760398070	7008625	SRX21396041	SRS18636199	SRA1694849	Whitehead Institute	Whitehead Institute	2	0.00245	0.0	0.00047	0.0	0.99803	1.0	0.58536		52	255	T	T	mates < 9% mapping rate	illumina	hiseq_era	unknown	poly_a	unknown	bulk	bulk	bulk	United States	2023-08-17	Multi-stage	Embryo	Embryo Imprecise	All anatomical structures
25186	25186	SRR25670733	SRX21396040	SRS18636198	SRP455680	PRJNA1006406	Control of polyA tail length and translation in vertebrate oocytes and early embryos	GSE241107	Other	During oocyte maturation and early embryonic development polyA tail lengths strongly influence mRNA translation. However how tail lengths are controlled at different developmental stages has been unclear. Here we performed tail length and translational profiling of mRNA reporter libraries each with > 10 million three prime UTR sequence variants in frog oocytes and embryos and fish embryos. These analyses revealed that the UUUUA motif specifies cytoplasmic polyadenylation and identified diverse context features that modulate the activity of this 5 mer. Additional sequence motifs drive stage specific deadenylation in embryos and UUUUA and C rich motifs drive tail length independent translational repression in oocytes. A neural network model accurately predicts tail length change during oocyte maturation in frogs mice and humans. Analyses of human sequence variants showed that those predicted to disrupt tail length control have been under negative selection implying that our insights into control of polyA tail length and translation have implications for human health and fertility. Overall design: Sythetic mRNA reporter libraries with random three prime UTR sequences under four different sequence contexts were injected into frog oocytes and embryos and fish embryos. PolyA tail lengths were measured to at different developmental stages to examine sequence motifs that caused tail length changes. At the same time polyA tail lengths of endogenous mRNAs from frog oocytes and embryos fish embryos and mouse oocytes were measured to investiage how their tail lengths were controlled. Please note that sample titles have been updated on Jan 6 2024.				Fish embryo mRNA sphere PAL seq v4	GSM7716869		source name:embryo\|tissue:embryo\|treatment:N1\|geo loc name:missing\|collection date:missing	Fish embryo mRNA sphere PAL seq v4	For gene specific tail seq of mRNA reporters data were processed with a custom script available at https://github.com/coffeebond/MPRA tail seq. For PAL seq v3 or v4 reads were trimmed with cutadapt v3.7 with the parameters “ m 15 quality base=64 q 20 20 match read wildcards e 0.05 a NNNNATCTCGTATGCCGTCTTCTGCTTG O 7”. The trimmed reads were mapped using STAR v2.7.1a to the reference database containing the genomic sequences of the organism from which the mRNAs were obtained the genomic sequences of humans or fish depending on which spike in RNAs were used and the sequences of the polyA standards generated previously with the parameters “ runThreadN 16 runMode alignReads outFilterMultimapNmax 1 outReadsUnmapped Fastx outFilterType BySJout outSAMattributes All outSAMtype BAM Unsorted SortedByCoordinate”. Uniquely mapped read were furhter processed with a custom script available at https://github.com/coffeebond/PAL seq. Assembly: Homo sapiens: GRCh38.p7 primary assembly. Mus muculus: GRCh38.p4 primary assembly. Xenopus laevis: v10.1 assembly. Danio rerio: GRCz11 assembly. Supplementary files format and content: For gene specific tail seq of mRNA reporters tab delimited text files with columns indicating 1 sequence of the variable region; 2 median tail length; 3 number of reads; 4–7 tail lengths at quantile 10 25 75 and 90. Supplementary files format and content: For PAL seq v3 or v4 tab delimited files with columns indicating 1 gene ID or polyA site ID; 2 read cluster ID; 3 tail length Library strategy: PAL seq v4	embryo		Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5 100 mM KCl 5 mM MgCl2 1% [v/v] Triton X 100 100 µg/ml cycloheximide cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer] and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr denuded with 3 mg/ml hyaluronidase for 2 min washed and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases but for the N60 library injected into fish embryos and frog oocytes the N37 PAS N17 library injected into fish embryos and frog oocytes and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009 8 pmol KXSH010 and 2x SSC 0.3 M NaCl 30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher 65002 and incubated for 20 min at 23°C on a thermal mixer shaking with 15 sec on and 1 min 45 sec off. The superna…		tissue:embryo\|treatment:N1	GSM7716869	GSM7716869: Fish embryo mRNA sphere PAL seq v4; Danio rerio; OTHER	GSM7716869 r1	GSM7716869	1	Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5 100 mM KCl 5 mM MgCl2 1% [v/v] Triton X 100 100 µg/ml cycloheximide cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer] and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr denuded with 3 mg/ml hyaluronidase for 2 min washed and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases but for the N60 library injected into fish embryos and frog oocytes the N37 PAS N17 library injected into fish embryos and frog oocytes and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009 8 pmol KXSH010 and 2x SSC 0.3 M NaCl 30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher 65002 and incubated for 20 min at 23°C on a thermal mixer shaking with 15 sec on and 1 min 45 sec off. The superna…	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	Illumina HiSeq 2500	SRP455680			Fish_embryo_mRNA_sphere_PAL_seq_v4_rep1_raw_read1.fastq.gz Fish_embryo_mRNA_sphere_PAL_seq_v4_rep1_raw_read2.fastq.gz	fastq fastq	3211687254.0	10461522.0	GSM7716869 r1	0:52 1:255	A:813560400;C:775326762;G:854563477;T:759161458;N:9075157	52	255			813560400	775326762	854563477	759161458	9075157	SRX21396040	SRS18636198	SRA1694849	Whitehead Institute	Whitehead Institute	2	0.00088	0.43191	0.0004	0.01556	0.99916	0.99961	0.55769	1.0	52	255	T	B	mate1 technical by mapping diff	illumina	hiseq_era	unknown	poly_a	unknown	bulk	bulk	bulk	United States	2023-08-17	Multi-stage	Embryo	Embryo Imprecise	All anatomical structures
25187	25187	SRR25670734	SRX21396040	SRS18636198	SRP455680	PRJNA1006406	Control of polyA tail length and translation in vertebrate oocytes and early embryos	GSE241107	Other	During oocyte maturation and early embryonic development polyA tail lengths strongly influence mRNA translation. However how tail lengths are controlled at different developmental stages has been unclear. Here we performed tail length and translational profiling of mRNA reporter libraries each with > 10 million three prime UTR sequence variants in frog oocytes and embryos and fish embryos. These analyses revealed that the UUUUA motif specifies cytoplasmic polyadenylation and identified diverse context features that modulate the activity of this 5 mer. Additional sequence motifs drive stage specific deadenylation in embryos and UUUUA and C rich motifs drive tail length independent translational repression in oocytes. A neural network model accurately predicts tail length change during oocyte maturation in frogs mice and humans. Analyses of human sequence variants showed that those predicted to disrupt tail length control have been under negative selection implying that our insights into control of polyA tail length and translation have implications for human health and fertility. Overall design: Sythetic mRNA reporter libraries with random three prime UTR sequences under four different sequence contexts were injected into frog oocytes and embryos and fish embryos. PolyA tail lengths were measured to at different developmental stages to examine sequence motifs that caused tail length changes. At the same time polyA tail lengths of endogenous mRNAs from frog oocytes and embryos fish embryos and mouse oocytes were measured to investiage how their tail lengths were controlled. Please note that sample titles have been updated on Jan 6 2024.				Fish embryo mRNA sphere PAL seq v4	GSM7716869		source name:embryo\|tissue:embryo\|treatment:N1\|geo loc name:missing\|collection date:missing	Fish embryo mRNA sphere PAL seq v4	For gene specific tail seq of mRNA reporters data were processed with a custom script available at https://github.com/coffeebond/MPRA tail seq. For PAL seq v3 or v4 reads were trimmed with cutadapt v3.7 with the parameters “ m 15 quality base=64 q 20 20 match read wildcards e 0.05 a NNNNATCTCGTATGCCGTCTTCTGCTTG O 7”. The trimmed reads were mapped using STAR v2.7.1a to the reference database containing the genomic sequences of the organism from which the mRNAs were obtained the genomic sequences of humans or fish depending on which spike in RNAs were used and the sequences of the polyA standards generated previously with the parameters “ runThreadN 16 runMode alignReads outFilterMultimapNmax 1 outReadsUnmapped Fastx outFilterType BySJout outSAMattributes All outSAMtype BAM Unsorted SortedByCoordinate”. Uniquely mapped read were furhter processed with a custom script available at https://github.com/coffeebond/PAL seq. Assembly: Homo sapiens: GRCh38.p7 primary assembly. Mus muculus: GRCh38.p4 primary assembly. Xenopus laevis: v10.1 assembly. Danio rerio: GRCz11 assembly. Supplementary files format and content: For gene specific tail seq of mRNA reporters tab delimited text files with columns indicating 1 sequence of the variable region; 2 median tail length; 3 number of reads; 4–7 tail lengths at quantile 10 25 75 and 90. Supplementary files format and content: For PAL seq v3 or v4 tab delimited files with columns indicating 1 gene ID or polyA site ID; 2 read cluster ID; 3 tail length Library strategy: PAL seq v4	embryo		Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5 100 mM KCl 5 mM MgCl2 1% [v/v] Triton X 100 100 µg/ml cycloheximide cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer] and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr denuded with 3 mg/ml hyaluronidase for 2 min washed and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases but for the N60 library injected into fish embryos and frog oocytes the N37 PAS N17 library injected into fish embryos and frog oocytes and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009 8 pmol KXSH010 and 2x SSC 0.3 M NaCl 30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher 65002 and incubated for 20 min at 23°C on a thermal mixer shaking with 15 sec on and 1 min 45 sec off. The superna…		tissue:embryo\|treatment:N1	GSM7716869	GSM7716869: Fish embryo mRNA sphere PAL seq v4; Danio rerio; OTHER	GSM7716869 r1	GSM7716869	1	Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5 100 mM KCl 5 mM MgCl2 1% [v/v] Triton X 100 100 µg/ml cycloheximide cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer] and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr denuded with 3 mg/ml hyaluronidase for 2 min washed and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases but for the N60 library injected into fish embryos and frog oocytes the N37 PAS N17 library injected into fish embryos and frog oocytes and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009 8 pmol KXSH010 and 2x SSC 0.3 M NaCl 30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher 65002 and incubated for 20 min at 23°C on a thermal mixer shaking with 15 sec on and 1 min 45 sec off. The superna…	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	Illumina HiSeq 2500	SRP455680			Fish_embryo_mRNA_sphere_PAL_seq_v4_rep2_raw_read1.fastq.gz Fish_embryo_mRNA_sphere_PAL_seq_v4_rep2_raw_read2.fastq.gz	fastq fastq	3179916131.0	10358033.0	GSM7716869 r2	0:52 1:255	A:771755667;C:804256300;G:883166175;T:714242073;N:6495916	52	255			771755667	804256300	883166175	714242073	6495916	SRX21396040	SRS18636198	SRA1694849	Whitehead Institute	Whitehead Institute	2	0.00186	0.0	0.00088	0.0	0.99862	1.0	0.64705		52	255	T	T	mates < 9% mapping rate	illumina	hiseq_era	unknown	poly_a	unknown	bulk	bulk	bulk	United States	2023-08-17	Multi-stage	Embryo	Embryo Imprecise	All anatomical structures
25190	25190	SRR25670737	SRX21396038	SRS18636196	SRP455680	PRJNA1006406	Control of polyA tail length and translation in vertebrate oocytes and early embryos	GSE241107	Other	During oocyte maturation and early embryonic development polyA tail lengths strongly influence mRNA translation. However how tail lengths are controlled at different developmental stages has been unclear. Here we performed tail length and translational profiling of mRNA reporter libraries each with > 10 million three prime UTR sequence variants in frog oocytes and embryos and fish embryos. These analyses revealed that the UUUUA motif specifies cytoplasmic polyadenylation and identified diverse context features that modulate the activity of this 5 mer. Additional sequence motifs drive stage specific deadenylation in embryos and UUUUA and C rich motifs drive tail length independent translational repression in oocytes. A neural network model accurately predicts tail length change during oocyte maturation in frogs mice and humans. Analyses of human sequence variants showed that those predicted to disrupt tail length control have been under negative selection implying that our insights into control of polyA tail length and translation have implications for human health and fertility. Overall design: Sythetic mRNA reporter libraries with random three prime UTR sequences under four different sequence contexts were injected into frog oocytes and embryos and fish embryos. PolyA tail lengths were measured to at different developmental stages to examine sequence motifs that caused tail length changes. At the same time polyA tail lengths of endogenous mRNAs from frog oocytes and embryos fish embryos and mouse oocytes were measured to investiage how their tail lengths were controlled. Please note that sample titles have been updated on Jan 6 2024.				Fish embryo mRNA 128cell PAL seq v4	GSM7716867		source name:embryo\|tissue:embryo\|treatment:N1\|geo loc name:missing\|collection date:missing	Fish embryo mRNA 128cell PAL seq v4	For gene specific tail seq of mRNA reporters data were processed with a custom script available at https://github.com/coffeebond/MPRA tail seq. For PAL seq v3 or v4 reads were trimmed with cutadapt v3.7 with the parameters “ m 15 quality base=64 q 20 20 match read wildcards e 0.05 a NNNNATCTCGTATGCCGTCTTCTGCTTG O 7”. The trimmed reads were mapped using STAR v2.7.1a to the reference database containing the genomic sequences of the organism from which the mRNAs were obtained the genomic sequences of humans or fish depending on which spike in RNAs were used and the sequences of the polyA standards generated previously with the parameters “ runThreadN 16 runMode alignReads outFilterMultimapNmax 1 outReadsUnmapped Fastx outFilterType BySJout outSAMattributes All outSAMtype BAM Unsorted SortedByCoordinate”. Uniquely mapped read were furhter processed with a custom script available at https://github.com/coffeebond/PAL seq. Assembly: Homo sapiens: GRCh38.p7 primary assembly. Mus muculus: GRCh38.p4 primary assembly. Xenopus laevis: v10.1 assembly. Danio rerio: GRCz11 assembly. Supplementary files format and content: For gene specific tail seq of mRNA reporters tab delimited text files with columns indicating 1 sequence of the variable region; 2 median tail length; 3 number of reads; 4–7 tail lengths at quantile 10 25 75 and 90. Supplementary files format and content: For PAL seq v3 or v4 tab delimited files with columns indicating 1 gene ID or polyA site ID; 2 read cluster ID; 3 tail length Library strategy: PAL seq v4	embryo		Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5 100 mM KCl 5 mM MgCl2 1% [v/v] Triton X 100 100 µg/ml cycloheximide cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer] and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr denuded with 3 mg/ml hyaluronidase for 2 min washed and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases but for the N60 library injected into fish embryos and frog oocytes the N37 PAS N17 library injected into fish embryos and frog oocytes and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009 8 pmol KXSH010 and 2x SSC 0.3 M NaCl 30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher 65002 and incubated for 20 min at 23°C on a thermal mixer shaking with 15 sec on and 1 min 45 sec off. The superna…		tissue:embryo\|treatment:N1	GSM7716867	GSM7716867: Fish embryo mRNA 128cell PAL seq v4; Danio rerio; OTHER	GSM7716867 r1	GSM7716867	1	Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5 100 mM KCl 5 mM MgCl2 1% [v/v] Triton X 100 100 µg/ml cycloheximide cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer] and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr denuded with 3 mg/ml hyaluronidase for 2 min washed and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases but for the N60 library injected into fish embryos and frog oocytes the N37 PAS N17 library injected into fish embryos and frog oocytes and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009 8 pmol KXSH010 and 2x SSC 0.3 M NaCl 30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher 65002 and incubated for 20 min at 23°C on a thermal mixer shaking with 15 sec on and 1 min 45 sec off. The superna…	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	Illumina HiSeq 2500	SRP455680			Fish_embryo_mRNA_128cell_PAL_seq_v4_rep1_raw_read1.fastq.gz Fish_embryo_mRNA_128cell_PAL_seq_v4_rep1_raw_read2.fastq.gz	fastq fastq	2104868443.0	6856249.0	GSM7716867 r1	0:52 1:255	A:539122676;C:505234642;G:558793048;T:495876292;N:5841785	52	255			539122676	505234642	558793048	495876292	5841785	SRX21396038	SRS18636196	SRA1694849	Whitehead Institute	Whitehead Institute	2	0.00035	0.29379	7e-05	0.01129	0.99949	0.99971	0.55882	0.79591	52	255	T	B	mate1 technical by mapping diff	illumina	hiseq_era	unknown	poly_a	unknown	bulk	bulk	bulk	United States	2023-08-17	Multi-stage	Embryo	Embryo Imprecise	All anatomical structures
25191	25191	SRR25670738	SRX21396038	SRS18636196	SRP455680	PRJNA1006406	Control of polyA tail length and translation in vertebrate oocytes and early embryos	GSE241107	Other	During oocyte maturation and early embryonic development polyA tail lengths strongly influence mRNA translation. However how tail lengths are controlled at different developmental stages has been unclear. Here we performed tail length and translational profiling of mRNA reporter libraries each with > 10 million three prime UTR sequence variants in frog oocytes and embryos and fish embryos. These analyses revealed that the UUUUA motif specifies cytoplasmic polyadenylation and identified diverse context features that modulate the activity of this 5 mer. Additional sequence motifs drive stage specific deadenylation in embryos and UUUUA and C rich motifs drive tail length independent translational repression in oocytes. A neural network model accurately predicts tail length change during oocyte maturation in frogs mice and humans. Analyses of human sequence variants showed that those predicted to disrupt tail length control have been under negative selection implying that our insights into control of polyA tail length and translation have implications for human health and fertility. Overall design: Sythetic mRNA reporter libraries with random three prime UTR sequences under four different sequence contexts were injected into frog oocytes and embryos and fish embryos. PolyA tail lengths were measured to at different developmental stages to examine sequence motifs that caused tail length changes. At the same time polyA tail lengths of endogenous mRNAs from frog oocytes and embryos fish embryos and mouse oocytes were measured to investiage how their tail lengths were controlled. Please note that sample titles have been updated on Jan 6 2024.				Fish embryo mRNA 128cell PAL seq v4	GSM7716867		source name:embryo\|tissue:embryo\|treatment:N1\|geo loc name:missing\|collection date:missing	Fish embryo mRNA 128cell PAL seq v4	For gene specific tail seq of mRNA reporters data were processed with a custom script available at https://github.com/coffeebond/MPRA tail seq. For PAL seq v3 or v4 reads were trimmed with cutadapt v3.7 with the parameters “ m 15 quality base=64 q 20 20 match read wildcards e 0.05 a NNNNATCTCGTATGCCGTCTTCTGCTTG O 7”. The trimmed reads were mapped using STAR v2.7.1a to the reference database containing the genomic sequences of the organism from which the mRNAs were obtained the genomic sequences of humans or fish depending on which spike in RNAs were used and the sequences of the polyA standards generated previously with the parameters “ runThreadN 16 runMode alignReads outFilterMultimapNmax 1 outReadsUnmapped Fastx outFilterType BySJout outSAMattributes All outSAMtype BAM Unsorted SortedByCoordinate”. Uniquely mapped read were furhter processed with a custom script available at https://github.com/coffeebond/PAL seq. Assembly: Homo sapiens: GRCh38.p7 primary assembly. Mus muculus: GRCh38.p4 primary assembly. Xenopus laevis: v10.1 assembly. Danio rerio: GRCz11 assembly. Supplementary files format and content: For gene specific tail seq of mRNA reporters tab delimited text files with columns indicating 1 sequence of the variable region; 2 median tail length; 3 number of reads; 4–7 tail lengths at quantile 10 25 75 and 90. Supplementary files format and content: For PAL seq v3 or v4 tab delimited files with columns indicating 1 gene ID or polyA site ID; 2 read cluster ID; 3 tail length Library strategy: PAL seq v4	embryo		Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5 100 mM KCl 5 mM MgCl2 1% [v/v] Triton X 100 100 µg/ml cycloheximide cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer] and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr denuded with 3 mg/ml hyaluronidase for 2 min washed and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases but for the N60 library injected into fish embryos and frog oocytes the N37 PAS N17 library injected into fish embryos and frog oocytes and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009 8 pmol KXSH010 and 2x SSC 0.3 M NaCl 30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher 65002 and incubated for 20 min at 23°C on a thermal mixer shaking with 15 sec on and 1 min 45 sec off. The superna…		tissue:embryo\|treatment:N1	GSM7716867	GSM7716867: Fish embryo mRNA 128cell PAL seq v4; Danio rerio; OTHER	GSM7716867 r1	GSM7716867	1	Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5 100 mM KCl 5 mM MgCl2 1% [v/v] Triton X 100 100 µg/ml cycloheximide cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer] and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr denuded with 3 mg/ml hyaluronidase for 2 min washed and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases but for the N60 library injected into fish embryos and frog oocytes the N37 PAS N17 library injected into fish embryos and frog oocytes and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009 8 pmol KXSH010 and 2x SSC 0.3 M NaCl 30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher 65002 and incubated for 20 min at 23°C on a thermal mixer shaking with 15 sec on and 1 min 45 sec off. The superna…	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	Illumina HiSeq 2500	SRP455680			Fish_embryo_mRNA_128cell_PAL_seq_v4_rep2_raw_read1.fastq.gz Fish_embryo_mRNA_128cell_PAL_seq_v4_rep2_raw_read2.fastq.gz	fastq fastq	3135806371.0	10214353.0	GSM7716867 r2	0:52 1:255	A:776612486;C:774126305;G:853951502;T:724788612;N:6327466	52	255			776612486	774126305	853951502	724788612	6327466	SRX21396038	SRS18636196	SRA1694849	Whitehead Institute	Whitehead Institute	2	0.00066	0.0	0.00011	0.0	0.99939	1.0	0.55737		52	255	T	T	mates < 9% mapping rate	illumina	hiseq_era	unknown	poly_a	unknown	bulk	bulk	bulk	United States	2023-08-17	Multi-stage	Embryo	Embryo Imprecise	All anatomical structures
25192	25192	SRR25670739	SRX21396037	SRS18636195	SRP455680	PRJNA1006406	Control of polyA tail length and translation in vertebrate oocytes and early embryos	GSE241107	Other	During oocyte maturation and early embryonic development polyA tail lengths strongly influence mRNA translation. However how tail lengths are controlled at different developmental stages has been unclear. Here we performed tail length and translational profiling of mRNA reporter libraries each with > 10 million three prime UTR sequence variants in frog oocytes and embryos and fish embryos. These analyses revealed that the UUUUA motif specifies cytoplasmic polyadenylation and identified diverse context features that modulate the activity of this 5 mer. Additional sequence motifs drive stage specific deadenylation in embryos and UUUUA and C rich motifs drive tail length independent translational repression in oocytes. A neural network model accurately predicts tail length change during oocyte maturation in frogs mice and humans. Analyses of human sequence variants showed that those predicted to disrupt tail length control have been under negative selection implying that our insights into control of polyA tail length and translation have implications for human health and fertility. Overall design: Sythetic mRNA reporter libraries with random three prime UTR sequences under four different sequence contexts were injected into frog oocytes and embryos and fish embryos. PolyA tail lengths were measured to at different developmental stages to examine sequence motifs that caused tail length changes. At the same time polyA tail lengths of endogenous mRNAs from frog oocytes and embryos fish embryos and mouse oocytes were measured to investiage how their tail lengths were controlled. Please note that sample titles have been updated on Jan 6 2024.				Fish embryo mRNA 8cell PAL seq v4	GSM7716866		source name:embryo\|tissue:embryo\|treatment:N1\|geo loc name:missing\|collection date:missing	Fish embryo mRNA 8cell PAL seq v4	For gene specific tail seq of mRNA reporters data were processed with a custom script available at https://github.com/coffeebond/MPRA tail seq. For PAL seq v3 or v4 reads were trimmed with cutadapt v3.7 with the parameters “ m 15 quality base=64 q 20 20 match read wildcards e 0.05 a NNNNATCTCGTATGCCGTCTTCTGCTTG O 7”. The trimmed reads were mapped using STAR v2.7.1a to the reference database containing the genomic sequences of the organism from which the mRNAs were obtained the genomic sequences of humans or fish depending on which spike in RNAs were used and the sequences of the polyA standards generated previously with the parameters “ runThreadN 16 runMode alignReads outFilterMultimapNmax 1 outReadsUnmapped Fastx outFilterType BySJout outSAMattributes All outSAMtype BAM Unsorted SortedByCoordinate”. Uniquely mapped read were furhter processed with a custom script available at https://github.com/coffeebond/PAL seq. Assembly: Homo sapiens: GRCh38.p7 primary assembly. Mus muculus: GRCh38.p4 primary assembly. Xenopus laevis: v10.1 assembly. Danio rerio: GRCz11 assembly. Supplementary files format and content: For gene specific tail seq of mRNA reporters tab delimited text files with columns indicating 1 sequence of the variable region; 2 median tail length; 3 number of reads; 4–7 tail lengths at quantile 10 25 75 and 90. Supplementary files format and content: For PAL seq v3 or v4 tab delimited files with columns indicating 1 gene ID or polyA site ID; 2 read cluster ID; 3 tail length Library strategy: PAL seq v4	embryo		Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5 100 mM KCl 5 mM MgCl2 1% [v/v] Triton X 100 100 µg/ml cycloheximide cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer] and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr denuded with 3 mg/ml hyaluronidase for 2 min washed and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases but for the N60 library injected into fish embryos and frog oocytes the N37 PAS N17 library injected into fish embryos and frog oocytes and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009 8 pmol KXSH010 and 2x SSC 0.3 M NaCl 30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher 65002 and incubated for 20 min at 23°C on a thermal mixer shaking with 15 sec on and 1 min 45 sec off. The superna…		tissue:embryo\|treatment:N1	GSM7716866	GSM7716866: Fish embryo mRNA 8cell PAL seq v4; Danio rerio; OTHER	GSM7716866 r1	GSM7716866	1	Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5 100 mM KCl 5 mM MgCl2 1% [v/v] Triton X 100 100 µg/ml cycloheximide cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer] and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr denuded with 3 mg/ml hyaluronidase for 2 min washed and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases but for the N60 library injected into fish embryos and frog oocytes the N37 PAS N17 library injected into fish embryos and frog oocytes and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009 8 pmol KXSH010 and 2x SSC 0.3 M NaCl 30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher 65002 and incubated for 20 min at 23°C on a thermal mixer shaking with 15 sec on and 1 min 45 sec off. The superna…	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	Illumina HiSeq 2500	SRP455680			Fish_embryo_mRNA_8cell_PAL_seq_v4_rep1_raw_read1.fastq.gz Fish_embryo_mRNA_8cell_PAL_seq_v4_rep1_raw_read2.fastq.gz	fastq fastq	2618998887.0	8530941.0	GSM7716866 r1	0:52 1:255	A:673066508;C:641446717;G:706546263;T:590452494;N:7486905	52	255			673066508	641446717	706546263	590452494	7486905	SRX21396037	SRS18636195	SRA1694849	Whitehead Institute	Whitehead Institute	2	0.0009	0.43244	0.00014	0.0054	0.99902	0.99967	0.54901	1.0	52	255	T	B	mate1 technical by mapping diff	illumina	hiseq_era	unknown	poly_a	unknown	bulk	bulk	bulk	United States	2023-08-17	Multi-stage	Embryo	Embryo Imprecise	All anatomical structures
25193	25193	SRR25670740	SRX21396037	SRS18636195	SRP455680	PRJNA1006406	Control of polyA tail length and translation in vertebrate oocytes and early embryos	GSE241107	Other	During oocyte maturation and early embryonic development polyA tail lengths strongly influence mRNA translation. However how tail lengths are controlled at different developmental stages has been unclear. Here we performed tail length and translational profiling of mRNA reporter libraries each with > 10 million three prime UTR sequence variants in frog oocytes and embryos and fish embryos. These analyses revealed that the UUUUA motif specifies cytoplasmic polyadenylation and identified diverse context features that modulate the activity of this 5 mer. Additional sequence motifs drive stage specific deadenylation in embryos and UUUUA and C rich motifs drive tail length independent translational repression in oocytes. A neural network model accurately predicts tail length change during oocyte maturation in frogs mice and humans. Analyses of human sequence variants showed that those predicted to disrupt tail length control have been under negative selection implying that our insights into control of polyA tail length and translation have implications for human health and fertility. Overall design: Sythetic mRNA reporter libraries with random three prime UTR sequences under four different sequence contexts were injected into frog oocytes and embryos and fish embryos. PolyA tail lengths were measured to at different developmental stages to examine sequence motifs that caused tail length changes. At the same time polyA tail lengths of endogenous mRNAs from frog oocytes and embryos fish embryos and mouse oocytes were measured to investiage how their tail lengths were controlled. Please note that sample titles have been updated on Jan 6 2024.				Fish embryo mRNA 8cell PAL seq v4	GSM7716866		source name:embryo\|tissue:embryo\|treatment:N1\|geo loc name:missing\|collection date:missing	Fish embryo mRNA 8cell PAL seq v4	For gene specific tail seq of mRNA reporters data were processed with a custom script available at https://github.com/coffeebond/MPRA tail seq. For PAL seq v3 or v4 reads were trimmed with cutadapt v3.7 with the parameters “ m 15 quality base=64 q 20 20 match read wildcards e 0.05 a NNNNATCTCGTATGCCGTCTTCTGCTTG O 7”. The trimmed reads were mapped using STAR v2.7.1a to the reference database containing the genomic sequences of the organism from which the mRNAs were obtained the genomic sequences of humans or fish depending on which spike in RNAs were used and the sequences of the polyA standards generated previously with the parameters “ runThreadN 16 runMode alignReads outFilterMultimapNmax 1 outReadsUnmapped Fastx outFilterType BySJout outSAMattributes All outSAMtype BAM Unsorted SortedByCoordinate”. Uniquely mapped read were furhter processed with a custom script available at https://github.com/coffeebond/PAL seq. Assembly: Homo sapiens: GRCh38.p7 primary assembly. Mus muculus: GRCh38.p4 primary assembly. Xenopus laevis: v10.1 assembly. Danio rerio: GRCz11 assembly. Supplementary files format and content: For gene specific tail seq of mRNA reporters tab delimited text files with columns indicating 1 sequence of the variable region; 2 median tail length; 3 number of reads; 4–7 tail lengths at quantile 10 25 75 and 90. Supplementary files format and content: For PAL seq v3 or v4 tab delimited files with columns indicating 1 gene ID or polyA site ID; 2 read cluster ID; 3 tail length Library strategy: PAL seq v4	embryo		Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5 100 mM KCl 5 mM MgCl2 1% [v/v] Triton X 100 100 µg/ml cycloheximide cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer] and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr denuded with 3 mg/ml hyaluronidase for 2 min washed and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases but for the N60 library injected into fish embryos and frog oocytes the N37 PAS N17 library injected into fish embryos and frog oocytes and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009 8 pmol KXSH010 and 2x SSC 0.3 M NaCl 30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher 65002 and incubated for 20 min at 23°C on a thermal mixer shaking with 15 sec on and 1 min 45 sec off. The superna…		tissue:embryo\|treatment:N1	GSM7716866	GSM7716866: Fish embryo mRNA 8cell PAL seq v4; Danio rerio; OTHER	GSM7716866 r1	GSM7716866	1	Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5 100 mM KCl 5 mM MgCl2 1% [v/v] Triton X 100 100 µg/ml cycloheximide cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer] and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr denuded with 3 mg/ml hyaluronidase for 2 min washed and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases but for the N60 library injected into fish embryos and frog oocytes the N37 PAS N17 library injected into fish embryos and frog oocytes and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009 8 pmol KXSH010 and 2x SSC 0.3 M NaCl 30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher 65002 and incubated for 20 min at 23°C on a thermal mixer shaking with 15 sec on and 1 min 45 sec off. The superna…	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	Illumina HiSeq 2500	SRP455680			Fish_embryo_mRNA_8cell_PAL_seq_v4_rep2_raw_read1.fastq.gz Fish_embryo_mRNA_8cell_PAL_seq_v4_rep2_raw_read2.fastq.gz	fastq fastq	3028839589.0	9865927.0	GSM7716866 r2	0:52 1:255	A:756368817;C:758868409;G:836949742;T:670545278;N:6107343	52	255			756368817	758868409	836949742	670545278	6107343	SRX21396037	SRS18636195	SRA1694849	Whitehead Institute	Whitehead Institute	2	0.00156	0.0	0.00026	0.0	0.99835	1.0	0.44791		52	255	T	T	mates < 9% mapping rate	illumina	hiseq_era	unknown	poly_a	unknown	bulk	bulk	bulk	United States	2023-08-17	Multi-stage	Embryo	Embryo Imprecise	All anatomical structures
32725	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 6–24 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"		pubmed:37546736		Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 2 3	GSM8327220		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32726	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 6–24 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"		pubmed:37546736		Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 2 3	GSM8327220		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32727	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 6–24 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"		pubmed:37546736		Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 2 3	GSM8327220		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32728	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 6–24 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"		pubmed:37546736		Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 2 3	GSM8327220		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32729	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 6–24 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"		pubmed:37546736		Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 1	GSM8327219		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32730	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 6–24 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"		pubmed:37546736		Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 1	GSM8327219		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32731	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 6–24 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"		pubmed:37546736		Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 1	GSM8327219		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32732	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 6–24 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"		pubmed:37546736		Perturbation experiment 3 24 hpf biological replicate 1 to 3 technical replicate 1	GSM8327219		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32733	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 6–24 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"		pubmed:37546736		Perturbation experiment 2 24 hpf biological replicate 3 technical replicate 1 to 4	GSM8327218		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32734	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 6–24 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"		pubmed:37546736		Perturbation experiment 2 24 hpf biological replicate 3 technical replicate 1 to 4	GSM8327218		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32735	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 6–24 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"		pubmed:37546736		Perturbation experiment 2 24 hpf biological replicate 3 technical replicate 1 to 4	GSM8327218		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32736	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 6–24 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"		pubmed:37546736		Perturbation experiment 2 24 hpf biological replicate 3 technical replicate 1 to 4	GSM8327218		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32737	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 6–24 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"		pubmed:37546736		Perturbation experiment 2 24 hpf biological replicate 2 technical replicate 1 to 6	GSM8327217		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32738	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 6–24 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"		pubmed:37546736		Perturbation experiment 2 24 hpf biological replicate 2 technical replicate 1 to 6	GSM8327217		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32739	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 6–24 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"		pubmed:37546736		Perturbation experiment 2 24 hpf biological replicate 2 technical replicate 1 to 6	GSM8327217		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32740	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 6–24 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"		pubmed:37546736		Perturbation experiment 2 24 hpf biological replicate 2 technical replicate 1 to 6	GSM8327217		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32741	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 6–24 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"		pubmed:37546736		Perturbation experiment 2 24 hpf biological replicate 1 technical replicate 1 to 4	GSM8327216		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32742	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 6–24 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"		pubmed:37546736		Perturbation experiment 2 24 hpf biological replicate 1 technical replicate 1 to 4	GSM8327216		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32743	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 6–24 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"		pubmed:37546736		Perturbation experiment 2 24 hpf biological replicate 1 technical replicate 1 to 4	GSM8327216		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32744	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 6–24 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"		pubmed:37546736		Perturbation experiment 2 24 hpf biological replicate 1 technical replicate 1 to 4	GSM8327216		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32745	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 6–24 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"		pubmed:37546736		Perturbation experiment 1 6 hpf 24 hpf biological replicate 3 technical replicate 1	GSM8327215		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32746	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 6–24 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"		pubmed:37546736		Perturbation experiment 1 6 hpf 24 hpf biological replicate 3 technical replicate 1	GSM8327215		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32747	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 6–24 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"		pubmed:37546736		Perturbation experiment 1 6 hpf 24 hpf biological replicate 3 technical replicate 1	GSM8327215		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32748	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 6–24 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"		pubmed:37546736		Perturbation experiment 1 6 hpf 24 hpf biological replicate 3 technical replicate 1	GSM8327215		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32749	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 6–24 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"		pubmed:37546736		Perturbation experiment 1 6 hpf 14 hpf biological replicate 1 technical replicate 1	GSM8327213		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32750	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 6–24 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"		pubmed:37546736		Perturbation experiment 1 6 hpf 14 hpf biological replicate 1 technical replicate 1	GSM8327213		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32751	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 6–24 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"		pubmed:37546736		Perturbation experiment 1 6 hpf 14 hpf biological replicate 1 technical replicate 1	GSM8327213		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32752	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 6–24 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"		pubmed:37546736		Perturbation experiment 1 6 hpf 14 hpf biological replicate 1 technical replicate 1	GSM8327213		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32753	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 6–24 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"		pubmed:37546736		Perturbation experiment 1 6 hpf 14 hpf biological replicate 1 technical replicate 1	GSM8327213		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32754	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 6–24 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"		pubmed:37546736		Perturbation experiment 1 6 hpf 14 hpf biological replicate 1 technical replicate 1	GSM8327213		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32755	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 6–24 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"		pubmed:37546736		Perturbation experiment 1 6 hpf 14 hpf biological replicate 1 technical replicate 1	GSM8327213		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32756	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 6–24 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"		pubmed:37546736		Perturbation experiment 1 6 hpf 14 hpf biological replicate 1 technical replicate 1	GSM8327213		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32757	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 6–24 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"		pubmed:37546736		Perturbation experiment 1 6 hpf 24 hpf biological replicate 2 technical replicate 1	GSM8327214		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32758	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 6–24 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"		pubmed:37546736		Perturbation experiment 1 6 hpf 24 hpf biological replicate 2 technical replicate 1	GSM8327214		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32759	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 6–24 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"		pubmed:37546736		Perturbation experiment 1 6 hpf 24 hpf biological replicate 2 technical replicate 1	GSM8327214		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32760	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 6–24 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"		pubmed:37546736		Perturbation experiment 1 6 hpf 24 hpf biological replicate 2 technical replicate 1	GSM8327214		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32761	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 6–24 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"		pubmed:37546736		Perturbation experiment 1 6 hpf 24 hpf biological replicate 2 technical replicate 1	GSM8327214		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32762	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 6–24 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"		pubmed:37546736		Perturbation experiment 1 6 hpf 24 hpf biological replicate 2 technical replicate 1	GSM8327214		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32763	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 6–24 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"		pubmed:37546736		Perturbation experiment 1 6 hpf 24 hpf biological replicate 2 technical replicate 1	GSM8327214		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32764	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 6–24 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"		pubmed:37546736		Perturbation experiment 1 6 hpf 24 hpf biological replicate 2 technical replicate 1	GSM8327214		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32765	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 6–24 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"		pubmed:37546736		Reference experiment 14 hpf 24 hpf biological replicate 2 and 3 technical replicate 2	GSM8327212		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32766	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 6–24 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"		pubmed:37546736		Reference experiment 14 hpf 24 hpf biological replicate 2 and 3 technical replicate 2	GSM8327212		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32767	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 6–24 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"		pubmed:37546736		Reference experiment 14 hpf 24 hpf biological replicate 2 and 3 technical replicate 2	GSM8327212		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32768	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 6–24 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"		pubmed:37546736		Reference experiment 14 hpf 24 hpf biological replicate 2 and 3 technical replicate 2	GSM8327212		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32769	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 6–24 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"		pubmed:37546736		Reference experiment 14 hpf 24 hpf biological replicate 1 technical replicate 2	GSM8327211		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32770	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 6–24 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"		pubmed:37546736		Reference experiment 14 hpf 24 hpf biological replicate 1 technical replicate 2	GSM8327211		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32771	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 6–24 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"		pubmed:37546736		Reference experiment 14 hpf 24 hpf biological replicate 1 technical replicate 2	GSM8327211		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32772	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 6–24 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"		pubmed:37546736		Reference experiment 14 hpf 24 hpf biological replicate 1 technical replicate 2	GSM8327211		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32773	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 6–24 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"		pubmed:37546736		Reference experiment 14 hpf 24 hpf biological replicate 2 and 3 technical replicate 1	GSM8327210		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32774	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 6–24 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"		pubmed:37546736		Reference experiment 14 hpf 24 hpf biological replicate 2 and 3 technical replicate 1	GSM8327210		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32775	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 6–24 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"		pubmed:37546736		Reference experiment 14 hpf 24 hpf biological replicate 2 and 3 technical replicate 1	GSM8327210		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32776	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 6–24 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"		pubmed:37546736		Reference experiment 14 hpf 24 hpf biological replicate 2 and 3 technical replicate 1	GSM8327210		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32777	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 6–24 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"		pubmed:37546736		Reference experiment 14 hpf 24 hpf biological replicate 1 technical replicate 1	GSM8327209		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32778	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 6–24 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"		pubmed:37546736		Reference experiment 14 hpf 24 hpf biological replicate 1 technical replicate 1	GSM8327209		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32779	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 6–24 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"		pubmed:37546736		Reference experiment 14 hpf 24 hpf biological replicate 1 technical replicate 1	GSM8327209		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32780	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 6–24 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"		pubmed:37546736		Reference experiment 14 hpf 24 hpf biological replicate 1 technical replicate 1	GSM8327209		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 ba…	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 – hydroxyurea Sigma Aldrich H8627 5G at 20 mM and aphidicolin Sigma Aldrich A0781 5MG at 150 µM 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 – 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed 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 µL FACSmax cell dissociation solution Genlantis T200100 for 14 hpf to 24 hpf embryos late time points. Cells were then filtered through a 40µm cell strainer mesh Fisher 352340 and centrifuged in a swinging bucket rotor at 200g for 1 minute early time points or 300g for 5 minutes late time points. Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium Sigma D1556 250ML. Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of 100 000 cells/mL before encapsulation. Library construction as detailed in Zilionis R. et al. 2016 Nature Protocols. Single cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP513754			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												B		usable mapping rate	illumina	nextseq	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-13	Multi-stage	Embryo	Whole Organism	All anatomical structures
32781	32781	SRR29411985	SRX24925451	SRS21630866	SRP513930	PRJNA1124008	Cell state transitions are decoupled from cell division during early embryo development [II]	GSE269848	Other	Paper abstract: As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: Design of experiment for this particular dataset: Forty tails from zebrafish embryos at 24 38 hpf and 48 hpf were collected. Cells were dissociated from these tails and multi seq tags were added to the cells for each time point. Cells from all time points were pooled and single cell sequencing was performed using Indrops. Four gene expression GEX libraries were prepared for transcriptomic information and four multiseq tag libraries TAG were prepared for reading out the multiseq tags. We provide the raw data .fastq files processed data both raw counts.tsv.gz and filtered by total UMI counts filtered.h5ad files and the entire filtered data post removing doublets as all data.h5ad. We also provide counts for multi seq tags example TAG 1.counts.csv. Information about pooling libraries and library indices is provided in Pooling Samples.xlsx. Only 24 hpf data was used for Kukreja et. al. 2024 and hence the cell state informa…		pubmed:37546736		24 38 hpf and 48 hpf tails	GSM8328864		source name:embryo tail\|tissue:embryo tail\|geo loc name:missing\|collection date:missing	24 38 hpf and 48 hpf tails	Reads were mapped onto the Zebrafish genome as described in Kukreja et al. 2024 manuscript. Multi seq barcodes for each sample were identified using a custom pipeline available here: https://github.com/AllonKleinLab/klunctions/. Barcode abundance was used to manually remove multiplet populations as well as assign cells to their appropriate sample. Data from only 24 hpf tails were used for the manuscript. We provide 8 FASTQ files 2 lanes x 4 reads per lane. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section "Single cell RNA seq Data preprocessing" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts for transcriptome tsv.gz: cell barcode gene names and raw counts for all cells Supplementary f…	embryo tail		Forty zebrafish tail samples collected at 24 38 hpf and 48 hpf were dissected between the yolk ball and extension and dissociated according to a modified version of the protocol described by Bresciani et al. 2018. Briefly the tails were dissociated with a mixture of DNaseI 20µg/mL Collagenase/Dispase 8 mg/mL and 0.25% Trypsin EDTA at 30.5°C for 15 minutes. The proteases were quenched with DMEM + 10% FBS. Samples were washed and resuspend in PBS. Cells from each timepoint were hashed with a unique lipid modified oligo using Multi seq. The barcoded samples were subsequently pooled washed with 1% BSA + PBS and resuspended in 0.1% BSA + 18% Optiprep in PBS at a final concentration of 300 000 cells/mL. Single cell transcriptomes were captured using inDrops. NGS libraries were prepared by the Single cell Core at Harvard Medical School and sequenced using an Illumina NovaSeq kit.		tissue:embryo tail	GSM8328864	GSM8328864: 24 38 hpf and 48 hpf tails; Danio rerio; OTHER	GSM8328864 r1	GSM8328864	1	Forty zebrafish tail samples collected at 24 38 hpf and 48 hpf were dissected between the yolk ball and extension and dissociated according to a modified version of the protocol described by Bresciani et al. 2018. Briefly the tails were dissociated with a mixture of DNaseI 20µg/mL Collagenase/Dispase 8 mg/mL and 0.25% Trypsin EDTA at 30.5°C for 15 minutes. The proteases were quenched with DMEM + 10% FBS. Samples were washed and resuspend in PBS. Cells from each timepoint were hashed with a unique lipid modified oligo using Multi seq. The barcoded samples were subsequently pooled washed with 1% BSA + PBS and resuspended in 0.1% BSA + 18% Optiprep in PBS at a final concentration of 300 000 cells/mL. Single cell transcriptomes were captured using inDrops. NGS libraries were prepared by the Single cell Core at Harvard Medical School and sequenced using an Illumina NovaSeq kit.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP513930			Undetermined_S0_L002_R1_001.fastq.gz Undetermined_S0_L002_R2_001.fastq.gz Undetermined_S0_L002_R3_001.fastq.gz Undetermined_S0_L002_R4_001.fastq.gz	fastq fastq fastq fastq	58603546956.0	505202991.0	GSM8328864 r1	0:86 1:8 2:8 3:14	A:13191204330;C:9162028793;G:9431065581;T:11662090182;N:1068340	86	8	8	14	13191204330	9162028793	9431065581	11662090182	1068340	SRX24925451	SRS21630866	SRA1899240	Harvard University	Harvard University												B		usable mapping rate	illumina	novaseq_era	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-14	Multi-stage	Embryo	Tail	Multi-system
32782	32782	SRR29411986	SRX24925451	SRS21630866	SRP513930	PRJNA1124008	Cell state transitions are decoupled from cell division during early embryo development [II]	GSE269848	Other	Paper abstract: As tissues develop cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However in the absence of cell division differentiation slows down in some cell types and cells exhibit global stress responses. While differentiation is robust to blocking cell division the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: Design of experiment for this particular dataset: Forty tails from zebrafish embryos at 24 38 hpf and 48 hpf were collected. Cells were dissociated from these tails and multi seq tags were added to the cells for each time point. Cells from all time points were pooled and single cell sequencing was performed using Indrops. Four gene expression GEX libraries were prepared for transcriptomic information and four multiseq tag libraries TAG were prepared for reading out the multiseq tags. We provide the raw data .fastq files processed data both raw counts.tsv.gz and filtered by total UMI counts filtered.h5ad files and the entire filtered data post removing doublets as all data.h5ad. We also provide counts for multi seq tags example TAG 1.counts.csv. Information about pooling libraries and library indices is provided in Pooling Samples.xlsx. Only 24 hpf data was used for Kukreja et. al. 2024 and hence the cell state informa…		pubmed:37546736		24 38 hpf and 48 hpf tails	GSM8328864		source name:embryo tail\|tissue:embryo tail\|geo loc name:missing\|collection date:missing	24 38 hpf and 48 hpf tails	Reads were mapped onto the Zebrafish genome as described in Kukreja et al. 2024 manuscript. Multi seq barcodes for each sample were identified using a custom pipeline available here: https://github.com/AllonKleinLab/klunctions/. Barcode abundance was used to manually remove multiplet populations as well as assign cells to their appropriate sample. Data from only 24 hpf tails were used for the manuscript. We provide 8 FASTQ files 2 lanes x 4 reads per lane. The read files from an inDrops run have the following content: * R1 001.fastq.gz : contains the three prime UTR cDNA read * R2 001.fastq.gz : contains the first half of the cell barcode * R3 001.fastq.gz : contains the library index for demultiplexing libraries * R4 001.fastq.gz : contains the second half of the barcode and the UMI. All subsequent processing steps from FASTQ files to count matrixes were performed using the custom pipeline for inDrops data analysis github.com/indrops. Single cell transcriptomes were barcoded using inDrops Klein et al Cell 2015. Standard transcriptome RNA seq libraries were processed as reported in Zilionis et al. Nature Protocol 2016 using inDrops v3 protocol. The transcriptome libraries were sequenced on reads Illumina NextSeq 500. Libraries used standard Illumina sequencing primers and 61 cycles for Read1 14 cycles for Read2 8 cycles each for IndexRead1 and IndexRead2. Raw fastq files was processed using inDrops.py pipeline github.com/indrops/indrops. Sequenced reads were mapped to a zebrafish reference transcriptome built from the zebrafish GRCz10 genome assembly Assembly Accession: GCF 000002035.5 using bowtie version 1.1.143. To obtain the final counts matrix used for data analysis total count filters were applied as described in Kukreja et. al. 2024 method section "Single cell RNA seq Data preprocessing" Assembly: GRCz10 genome assembly Assembly Accession: GCF 000002035.5 Supplementary files format and content: Raw counts for transcriptome tsv.gz: cell barcode gene names and raw counts for all cells Supplementary f…	embryo tail		Forty zebrafish tail samples collected at 24 38 hpf and 48 hpf were dissected between the yolk ball and extension and dissociated according to a modified version of the protocol described by Bresciani et al. 2018. Briefly the tails were dissociated with a mixture of DNaseI 20µg/mL Collagenase/Dispase 8 mg/mL and 0.25% Trypsin EDTA at 30.5°C for 15 minutes. The proteases were quenched with DMEM + 10% FBS. Samples were washed and resuspend in PBS. Cells from each timepoint were hashed with a unique lipid modified oligo using Multi seq. The barcoded samples were subsequently pooled washed with 1% BSA + PBS and resuspended in 0.1% BSA + 18% Optiprep in PBS at a final concentration of 300 000 cells/mL. Single cell transcriptomes were captured using inDrops. NGS libraries were prepared by the Single cell Core at Harvard Medical School and sequenced using an Illumina NovaSeq kit.		tissue:embryo tail	GSM8328864	GSM8328864: 24 38 hpf and 48 hpf tails; Danio rerio; OTHER	GSM8328864 r1	GSM8328864	1	Forty zebrafish tail samples collected at 24 38 hpf and 48 hpf were dissected between the yolk ball and extension and dissociated according to a modified version of the protocol described by Bresciani et al. 2018. Briefly the tails were dissociated with a mixture of DNaseI 20µg/mL Collagenase/Dispase 8 mg/mL and 0.25% Trypsin EDTA at 30.5°C for 15 minutes. The proteases were quenched with DMEM + 10% FBS. Samples were washed and resuspend in PBS. Cells from each timepoint were hashed with a unique lipid modified oligo using Multi seq. The barcoded samples were subsequently pooled washed with 1% BSA + PBS and resuspended in 0.1% BSA + 18% Optiprep in PBS at a final concentration of 300 000 cells/mL. Single cell transcriptomes were captured using inDrops. NGS libraries were prepared by the Single cell Core at Harvard Medical School and sequenced using an Illumina NovaSeq kit.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP513930			Undetermined_S0_L001_R1_001.fastq.gz Undetermined_S0_L001_R2_001.fastq.gz Undetermined_S0_L001_R3_001.fastq.gz Undetermined_S0_L001_R4_001.fastq.gz	fastq fastq fastq fastq	44072422880.0	379934680.0	GSM8328864 r2	0:86 1:8 2:8 3:14	A:9919784228;C:6894965731;G:7094749745;T:8764100624;N:782152	86	8	8	14	9919784228	6894965731	7094749745	8764100624	782152	SRX24925451	SRS21630866	SRA1899240	Harvard University	Harvard University												B		usable mapping rate	illumina	novaseq_era	unknown	other	trueseq	sc	single_cell_droplet	indrops	United States	2024-06-14	Multi-stage	Embryo	Tail	Multi-system
34614	34614	SRR32137213	SRX27483377	SRS23906281	SRP560121	PRJNA1216205	rbm24a is an organizer component of germ plasm to determine germ cell fate 202409 validation	GSE288109	Other	The formation of germ cells is a critical issue for the continuation of species. A large group of animals follow a preformation strategy to generate their primordial germ cells PGCs. They produce a set of localized maternal mRNAs and proteins to form phase separated germ plasm that functions as the PGC determinants but the mechanisms underlying the assembly of germ plasm is poorly understood. This study identifies Rbm24a as an enssential localized germ plasm protein component that controls the formation of large and functional germ plasm granules. Rbm24a is complexed with Buc and interacts with germ plasm mRNAs which determines the specific grasp of germ plasm mRNAs into the phase separated aggregates. Rbm24a absent granules fail to undergo kinesin dependent transport towards the cleavage furrows where small particles fuse into large ones. The loss of maternal rbm24a causes the complete degradation of germ plasm components and the disappearance of PGCs resulting in totally sterile animals. Our work establishes that Rbm24 is a critical nucleating organizer component of germ plasm highlighting an emerging common mechanism to read and recruit RNA component into phase separated condensates. Overall design: To comprehensively characterize the RNA binding landscape of Rbm24a in zebrafish we performed RIP seq analysis using rbm24a GFP knock in samples with IgG serving as the control.				Zebrafish RIP seq Rbm24a Knock in2 IP2	GSM8759069		source name:whole embryo\|tissue:whole embryo\|cell line:4 cell stage\|cell type:embryonic cell\|genotype:knock in\|geo loc name:missing\|collection date:missing	Zebrafish RIP seq Rbm24a Knock in2 IP2	Each library was amplified by a 15 cycle PCR and 150 bp paired end sequencing was subjected to Illumina Nova seq 6000 to obtain the raw data. Assembly: GRCz11 Supplementary files format and content: tab delimited text file includes raw counts for each Sample Supplementary files format and content: Bigwig file for each Sample	whole embryo		Total RNAs were extracted from Mrbm24a embryos at 4 cell sphere 24 hpf stage using TRIzol reagent Invitrogen and were precipitated by cold isopropanol 50% v/v in the presence of carrier glycogen 20 µg/each sample. 1 μg of immunoprecipitated RNA was utilized for library construction. Ribosomal RNA depletion was performed using RNase H based probes followed by magnetic bead purification. RNA fragmentation was achieved through magnesium catalyzed hydrolysis under elevated temperature 94°C for 6 min. First strand cDNA synthesis was initiated with random hexamer primers and reverse transcriptase followed by second strand synthesis using DNA polymerase I with dUTP incorporation for strand marking. Double stranded cDNA fragments underwent end repair and three prime adenylation using a single tube enzymatic reaction system. Illumina compatible adaptors with unique dual indexes were ligated to both ends of the DNA fragments. Size selection of adaptor ligated products 180 250 bp was performed using double sided SPRI bead purification. PCR amplification was conducted with P5/P7 primers for 12 cycles followed by AMPure XP bead purification to remove primer dimers. Library quality was verified by Agilent 2100 Bioanalyzer DNA High Sensitivity chip analysis. Indexed libraries were pooled in equimolar ratios and subjected to paired end sequencing 2×150 bp on an Illumina NovaSeq 6000 platform following the manufacturer's standardized workflow.	Zebrafish embryos were maintained in 1/3x Ringer's at 28.5C	tissue:whole embryo\|cell line:4 cell stage\|cell type:embryonic cell\|genotype:knock in	GSM8759069	GSM8759069: Zebrafish RIP seq Rbm24a Knock in2 IP2; Danio rerio; RIP Seq	GSM8759069 r1	GSM8759069	1	Total RNAs were extracted from Mrbm24a embryos at 4 cell sphere 24 hpf stage using TRIzol reagent Invitrogen and were precipitated by cold isopropanol 50% v/v in the presence of carrier glycogen 20 µg/each sample. 1 μg of immunoprecipitated RNA was utilized for library construction. Ribosomal RNA depletion was performed using RNase H based probes followed by magnetic bead purification. RNA fragmentation was achieved through magnesium catalyzed hydrolysis under elevated temperature 94°C for 6 min. First strand cDNA synthesis was initiated with random hexamer primers and reverse transcriptase followed by second strand synthesis using DNA polymerase I with dUTP incorporation for strand marking. Double stranded cDNA fragments underwent end repair and three prime adenylation using a single tube enzymatic reaction system. Illumina compatible adaptors with unique dual indexes were ligated to both ends of the DNA fragments. Size selection of adaptor ligated products 180 250 bp was performed using double sided SPRI bead purification. PCR amplification was conducted with P5/P7 primers for 12 cycles followed by AMPure XP bead purification to remove primer dimers. Library quality was verified by Agilent 2100 Bioanalyzer DNA High Sensitivity chip analysis. Indexed libraries were pooled in equimolar ratios and subjected to paired end sequencing 2×150 bp on an Illumina NovaSeq 6000 platform following the manufacturer's standardized workflow.	RIP-Seq	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP560121			RIP_IP2.R1.fq.gz RIP_IP2.R2.fq.gz	fastq fastq	18215080774.0	60314837.0	GSM8759069 r1	0:151 1:151	A:3824315591;C:5035112770;G:5644328190;T:3707941000;N:3383223	151	151			3824315591	5035112770	5644328190	3707941000	3383223	SRX27483377	SRS23906281	SRA2061743	Ang Li, College of Life Sciences, shandong university	Ang Li, College of Life Sciences, shandong university												B	B	mate2-mate1 similar by mapping diff	illumina	novaseq_era	unknown	random_priming	unknown	bulk	unknown	unknown	China	2025-01-27	Multi-stage	Embryo	Whole Organism	All anatomical structures
34615	34615	SRR32137214	SRX27483376	SRS23906280	SRP560121	PRJNA1216205	rbm24a is an organizer component of germ plasm to determine germ cell fate 202409 validation	GSE288109	Other	The formation of germ cells is a critical issue for the continuation of species. A large group of animals follow a preformation strategy to generate their primordial germ cells PGCs. They produce a set of localized maternal mRNAs and proteins to form phase separated germ plasm that functions as the PGC determinants but the mechanisms underlying the assembly of germ plasm is poorly understood. This study identifies Rbm24a as an enssential localized germ plasm protein component that controls the formation of large and functional germ plasm granules. Rbm24a is complexed with Buc and interacts with germ plasm mRNAs which determines the specific grasp of germ plasm mRNAs into the phase separated aggregates. Rbm24a absent granules fail to undergo kinesin dependent transport towards the cleavage furrows where small particles fuse into large ones. The loss of maternal rbm24a causes the complete degradation of germ plasm components and the disappearance of PGCs resulting in totally sterile animals. Our work establishes that Rbm24 is a critical nucleating organizer component of germ plasm highlighting an emerging common mechanism to read and recruit RNA component into phase separated condensates. Overall design: To comprehensively characterize the RNA binding landscape of Rbm24a in zebrafish we performed RIP seq analysis using rbm24a GFP knock in samples with IgG serving as the control.				Zebrafish RIP seq Rbm24a Knock in1 IP1	GSM8759068		source name:whole embryo\|tissue:whole embryo\|cell line:4 cell stage\|cell type:embryonic cell\|genotype:knock in\|geo loc name:missing\|collection date:missing	Zebrafish RIP seq Rbm24a Knock in1 IP1	Each library was amplified by a 15 cycle PCR and 150 bp paired end sequencing was subjected to Illumina Nova seq 6000 to obtain the raw data. Assembly: GRCz11 Supplementary files format and content: tab delimited text file includes raw counts for each Sample Supplementary files format and content: Bigwig file for each Sample	whole embryo		Total RNAs were extracted from Mrbm24a embryos at 4 cell sphere 24 hpf stage using TRIzol reagent Invitrogen and were precipitated by cold isopropanol 50% v/v in the presence of carrier glycogen 20 µg/each sample. 1 μg of immunoprecipitated RNA was utilized for library construction. Ribosomal RNA depletion was performed using RNase H based probes followed by magnetic bead purification. RNA fragmentation was achieved through magnesium catalyzed hydrolysis under elevated temperature 94°C for 6 min. First strand cDNA synthesis was initiated with random hexamer primers and reverse transcriptase followed by second strand synthesis using DNA polymerase I with dUTP incorporation for strand marking. Double stranded cDNA fragments underwent end repair and three prime adenylation using a single tube enzymatic reaction system. Illumina compatible adaptors with unique dual indexes were ligated to both ends of the DNA fragments. Size selection of adaptor ligated products 180 250 bp was performed using double sided SPRI bead purification. PCR amplification was conducted with P5/P7 primers for 12 cycles followed by AMPure XP bead purification to remove primer dimers. Library quality was verified by Agilent 2100 Bioanalyzer DNA High Sensitivity chip analysis. Indexed libraries were pooled in equimolar ratios and subjected to paired end sequencing 2×150 bp on an Illumina NovaSeq 6000 platform following the manufacturer's standardized workflow.	Zebrafish embryos were maintained in 1/3x Ringer's at 28.5C	tissue:whole embryo\|cell line:4 cell stage\|cell type:embryonic cell\|genotype:knock in	GSM8759068	GSM8759068: Zebrafish RIP seq Rbm24a Knock in1 IP1; Danio rerio; RIP Seq	GSM8759068 r1	GSM8759068	1	Total RNAs were extracted from Mrbm24a embryos at 4 cell sphere 24 hpf stage using TRIzol reagent Invitrogen and were precipitated by cold isopropanol 50% v/v in the presence of carrier glycogen 20 µg/each sample. 1 μg of immunoprecipitated RNA was utilized for library construction. Ribosomal RNA depletion was performed using RNase H based probes followed by magnetic bead purification. RNA fragmentation was achieved through magnesium catalyzed hydrolysis under elevated temperature 94°C for 6 min. First strand cDNA synthesis was initiated with random hexamer primers and reverse transcriptase followed by second strand synthesis using DNA polymerase I with dUTP incorporation for strand marking. Double stranded cDNA fragments underwent end repair and three prime adenylation using a single tube enzymatic reaction system. Illumina compatible adaptors with unique dual indexes were ligated to both ends of the DNA fragments. Size selection of adaptor ligated products 180 250 bp was performed using double sided SPRI bead purification. PCR amplification was conducted with P5/P7 primers for 12 cycles followed by AMPure XP bead purification to remove primer dimers. Library quality was verified by Agilent 2100 Bioanalyzer DNA High Sensitivity chip analysis. Indexed libraries were pooled in equimolar ratios and subjected to paired end sequencing 2×150 bp on an Illumina NovaSeq 6000 platform following the manufacturer's standardized workflow.	RIP-Seq	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP560121			RIP_IP1.R1.fq.gz RIP_IP1.R2.fq.gz	fastq fastq	22190472874.0	73478387.0	GSM8759068 r1	0:151 1:151	A:4585155428;C:6233832602;G:6882198251;T:4484977909;N:4308684	151	151			4585155428	6233832602	6882198251	4484977909	4308684	SRX27483376	SRS23906280	SRA2061743	Ang Li, College of Life Sciences, shandong university	Ang Li, College of Life Sciences, shandong university												B	B	mate2-mate1 similar by mapping diff	illumina	novaseq_era	unknown	random_priming	unknown	bulk	unknown	unknown	China	2025-01-27	Multi-stage	Embryo	Whole Organism	All anatomical structures
34616	34616	SRR32137215	SRX27483375	SRS23906279	SRP560121	PRJNA1216205	rbm24a is an organizer component of germ plasm to determine germ cell fate 202409 validation	GSE288109	Other	The formation of germ cells is a critical issue for the continuation of species. A large group of animals follow a preformation strategy to generate their primordial germ cells PGCs. They produce a set of localized maternal mRNAs and proteins to form phase separated germ plasm that functions as the PGC determinants but the mechanisms underlying the assembly of germ plasm is poorly understood. This study identifies Rbm24a as an enssential localized germ plasm protein component that controls the formation of large and functional germ plasm granules. Rbm24a is complexed with Buc and interacts with germ plasm mRNAs which determines the specific grasp of germ plasm mRNAs into the phase separated aggregates. Rbm24a absent granules fail to undergo kinesin dependent transport towards the cleavage furrows where small particles fuse into large ones. The loss of maternal rbm24a causes the complete degradation of germ plasm components and the disappearance of PGCs resulting in totally sterile animals. Our work establishes that Rbm24 is a critical nucleating organizer component of germ plasm highlighting an emerging common mechanism to read and recruit RNA component into phase separated condensates. Overall design: To comprehensively characterize the RNA binding landscape of Rbm24a in zebrafish we performed RIP seq analysis using rbm24a GFP knock in samples with IgG serving as the control.				Zebrafish RIP seq Rbm24a Knock in2 IgG2	GSM8759067		source name:whole embryo\|tissue:whole embryo\|cell line:4 cell stage\|cell type:embryonic cell\|genotype:knock in\|geo loc name:missing\|collection date:missing	Zebrafish RIP seq Rbm24a Knock in2 IgG2	Each library was amplified by a 15 cycle PCR and 150 bp paired end sequencing was subjected to Illumina Nova seq 6000 to obtain the raw data. Assembly: GRCz11 Supplementary files format and content: tab delimited text file includes raw counts for each Sample Supplementary files format and content: Bigwig file for each Sample	whole embryo		Total RNAs were extracted from Mrbm24a embryos at 4 cell sphere 24 hpf stage using TRIzol reagent Invitrogen and were precipitated by cold isopropanol 50% v/v in the presence of carrier glycogen 20 µg/each sample. 1 μg of immunoprecipitated RNA was utilized for library construction. Ribosomal RNA depletion was performed using RNase H based probes followed by magnetic bead purification. RNA fragmentation was achieved through magnesium catalyzed hydrolysis under elevated temperature 94°C for 6 min. First strand cDNA synthesis was initiated with random hexamer primers and reverse transcriptase followed by second strand synthesis using DNA polymerase I with dUTP incorporation for strand marking. Double stranded cDNA fragments underwent end repair and three prime adenylation using a single tube enzymatic reaction system. Illumina compatible adaptors with unique dual indexes were ligated to both ends of the DNA fragments. Size selection of adaptor ligated products 180 250 bp was performed using double sided SPRI bead purification. PCR amplification was conducted with P5/P7 primers for 12 cycles followed by AMPure XP bead purification to remove primer dimers. Library quality was verified by Agilent 2100 Bioanalyzer DNA High Sensitivity chip analysis. Indexed libraries were pooled in equimolar ratios and subjected to paired end sequencing 2×150 bp on an Illumina NovaSeq 6000 platform following the manufacturer's standardized workflow.	Zebrafish embryos were maintained in 1/3x Ringer's at 28.5C	tissue:whole embryo\|cell line:4 cell stage\|cell type:embryonic cell\|genotype:knock in	GSM8759067	GSM8759067: Zebrafish RIP seq Rbm24a Knock in2 IgG2; Danio rerio; RIP Seq	GSM8759067 r1	GSM8759067	1	Total RNAs were extracted from Mrbm24a embryos at 4 cell sphere 24 hpf stage using TRIzol reagent Invitrogen and were precipitated by cold isopropanol 50% v/v in the presence of carrier glycogen 20 µg/each sample. 1 μg of immunoprecipitated RNA was utilized for library construction. Ribosomal RNA depletion was performed using RNase H based probes followed by magnetic bead purification. RNA fragmentation was achieved through magnesium catalyzed hydrolysis under elevated temperature 94°C for 6 min. First strand cDNA synthesis was initiated with random hexamer primers and reverse transcriptase followed by second strand synthesis using DNA polymerase I with dUTP incorporation for strand marking. Double stranded cDNA fragments underwent end repair and three prime adenylation using a single tube enzymatic reaction system. Illumina compatible adaptors with unique dual indexes were ligated to both ends of the DNA fragments. Size selection of adaptor ligated products 180 250 bp was performed using double sided SPRI bead purification. PCR amplification was conducted with P5/P7 primers for 12 cycles followed by AMPure XP bead purification to remove primer dimers. Library quality was verified by Agilent 2100 Bioanalyzer DNA High Sensitivity chip analysis. Indexed libraries were pooled in equimolar ratios and subjected to paired end sequencing 2×150 bp on an Illumina NovaSeq 6000 platform following the manufacturer's standardized workflow.	RIP-Seq	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP560121			RIP_IgG2.R1.fq.gz RIP_IgG2.R2.fq.gz	fastq fastq	18588838088.0	61552444.0	GSM8759067 r1	0:151 1:151	A:3864885070;C:5250483318;G:5713729791;T:3756127723;N:3612186	151	151			3864885070	5250483318	5713729791	3756127723	3612186	SRX27483375	SRS23906279	SRA2061743	Ang Li, College of Life Sciences, shandong university	Ang Li, College of Life Sciences, shandong university												B	T	mate2 technical by mapping diff	illumina	novaseq_era	unknown	random_priming	unknown	bulk	unknown	unknown	China	2025-01-27	Multi-stage	Embryo	Whole Organism	All anatomical structures
34617	34617	SRR32137216	SRX27483374	SRS23906278	SRP560121	PRJNA1216205	rbm24a is an organizer component of germ plasm to determine germ cell fate 202409 validation	GSE288109	Other	The formation of germ cells is a critical issue for the continuation of species. A large group of animals follow a preformation strategy to generate their primordial germ cells PGCs. They produce a set of localized maternal mRNAs and proteins to form phase separated germ plasm that functions as the PGC determinants but the mechanisms underlying the assembly of germ plasm is poorly understood. This study identifies Rbm24a as an enssential localized germ plasm protein component that controls the formation of large and functional germ plasm granules. Rbm24a is complexed with Buc and interacts with germ plasm mRNAs which determines the specific grasp of germ plasm mRNAs into the phase separated aggregates. Rbm24a absent granules fail to undergo kinesin dependent transport towards the cleavage furrows where small particles fuse into large ones. The loss of maternal rbm24a causes the complete degradation of germ plasm components and the disappearance of PGCs resulting in totally sterile animals. Our work establishes that Rbm24 is a critical nucleating organizer component of germ plasm highlighting an emerging common mechanism to read and recruit RNA component into phase separated condensates. Overall design: To comprehensively characterize the RNA binding landscape of Rbm24a in zebrafish we performed RIP seq analysis using rbm24a GFP knock in samples with IgG serving as the control.				Zebrafish RIP seq Rbm24a Knock in1 IgG1	GSM8759066		source name:whole embryo\|tissue:whole embryo\|cell line:4 cell stage\|cell type:embryonic cell\|genotype:knock in\|geo loc name:missing\|collection date:missing	Zebrafish RIP seq Rbm24a Knock in1 IgG1	Each library was amplified by a 15 cycle PCR and 150 bp paired end sequencing was subjected to Illumina Nova seq 6000 to obtain the raw data. Assembly: GRCz11 Supplementary files format and content: tab delimited text file includes raw counts for each Sample Supplementary files format and content: Bigwig file for each Sample	whole embryo		Total RNAs were extracted from Mrbm24a embryos at 4 cell sphere 24 hpf stage using TRIzol reagent Invitrogen and were precipitated by cold isopropanol 50% v/v in the presence of carrier glycogen 20 µg/each sample. 1 μg of immunoprecipitated RNA was utilized for library construction. Ribosomal RNA depletion was performed using RNase H based probes followed by magnetic bead purification. RNA fragmentation was achieved through magnesium catalyzed hydrolysis under elevated temperature 94°C for 6 min. First strand cDNA synthesis was initiated with random hexamer primers and reverse transcriptase followed by second strand synthesis using DNA polymerase I with dUTP incorporation for strand marking. Double stranded cDNA fragments underwent end repair and three prime adenylation using a single tube enzymatic reaction system. Illumina compatible adaptors with unique dual indexes were ligated to both ends of the DNA fragments. Size selection of adaptor ligated products 180 250 bp was performed using double sided SPRI bead purification. PCR amplification was conducted with P5/P7 primers for 12 cycles followed by AMPure XP bead purification to remove primer dimers. Library quality was verified by Agilent 2100 Bioanalyzer DNA High Sensitivity chip analysis. Indexed libraries were pooled in equimolar ratios and subjected to paired end sequencing 2×150 bp on an Illumina NovaSeq 6000 platform following the manufacturer's standardized workflow.	Zebrafish embryos were maintained in 1/3x Ringer's at 28.5C	tissue:whole embryo\|cell line:4 cell stage\|cell type:embryonic cell\|genotype:knock in	GSM8759066	GSM8759066: Zebrafish RIP seq Rbm24a Knock in1 IgG1; Danio rerio; RIP Seq	GSM8759066 r1	GSM8759066	1	Total RNAs were extracted from Mrbm24a embryos at 4 cell sphere 24 hpf stage using TRIzol reagent Invitrogen and were precipitated by cold isopropanol 50% v/v in the presence of carrier glycogen 20 µg/each sample. 1 μg of immunoprecipitated RNA was utilized for library construction. Ribosomal RNA depletion was performed using RNase H based probes followed by magnetic bead purification. RNA fragmentation was achieved through magnesium catalyzed hydrolysis under elevated temperature 94°C for 6 min. First strand cDNA synthesis was initiated with random hexamer primers and reverse transcriptase followed by second strand synthesis using DNA polymerase I with dUTP incorporation for strand marking. Double stranded cDNA fragments underwent end repair and three prime adenylation using a single tube enzymatic reaction system. Illumina compatible adaptors with unique dual indexes were ligated to both ends of the DNA fragments. Size selection of adaptor ligated products 180 250 bp was performed using double sided SPRI bead purification. PCR amplification was conducted with P5/P7 primers for 12 cycles followed by AMPure XP bead purification to remove primer dimers. Library quality was verified by Agilent 2100 Bioanalyzer DNA High Sensitivity chip analysis. Indexed libraries were pooled in equimolar ratios and subjected to paired end sequencing 2×150 bp on an Illumina NovaSeq 6000 platform following the manufacturer's standardized workflow.	RIP-Seq	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP560121			RIP_IgG1.R1.fq.gz RIP_IgG1.R2.fq.gz	fastq fastq	18008086350.0	59629425.0	GSM8759066 r1	0:151 1:151	A:3736851298;C:5091099145;G:5529269310;T:3647429389;N:3437208	151	151			3736851298	5091099145	5529269310	3647429389	3437208	SRX27483374	SRS23906278	SRA2061743	Ang Li, College of Life Sciences, shandong university	Ang Li, College of Life Sciences, shandong university												B	T	mate2 technical by mapping diff	illumina	novaseq_era	unknown	random_priming	unknown	bulk	unknown	unknown	China	2025-01-27	Multi-stage	Embryo	Whole Organism	All anatomical structures
36053	36053	SRR33444765	SRX28685529	SRS24952431	SRP583274	PRJNA1259243	Structure Dependent Developmental Toxicity of Alkyl Substituted Naphthalenes	GSE296410	Transcriptome Analysis	Naphthalene and its alkyl substituted derivatives are among the most abundant polycyclic aromatic hydrocarbons PAHs in environmental and human exposure studies yet their developmental toxicity and mode of action remain poorly understood due to challenges in testing of semi volatile compounds. This study developed a vial based high through put method to effectively assess the activity of naphthalenes and a set of 24 alkyl substituted naphthalenes. Early life stage zebrafish were exposed to a concentration series of each chemical 0 50 µM in rotating sealed glass vials to minimize volatilization. Benchmark concentration BMC50 values were calculated for morphological endpoints and lowest effect levels were determined for behavioral effects. The data were assessed for evidence of a narcotic mode of action using body burden measurements for select chemicals and logKow modeling. Targeted transcriptomics at a single concentration and timepoint as well as in silico molecular docking were conducted to generate mode of action hypothesis. The vial method enabled detection of highly variable developmental toxicity not previously observed using standard 96 well plate exposures. LogKow and body burden were poor predictors of toxicity suggesting a non narcotic mode of action. Transcriptomic analysis revealed a limited but notable set of differentially expressed genes with evidence for the disruption of glucocorticoid signaling pathways. Molecular docking identified potential protein targets e.g. CYP1A2 NT5E FOLR1 that may mediate observed effects. This study demonstrates the importance of appropriate exposure methods for semi volatile compounds reveals structure dependent toxicity among alkyl substituted naphthalenes and provides a foundation for further mechanistic studies and improved risk assessment of alkyl substituted PAHs. Overall design: Zebrafish were exposed to 25 chemicals naphthalene and 24 alkyl sustituted naphthalenes using a vial exposure method. Embryos were exposed to 20 uM of each chemical in groups of…				1 8 dimethylnaphthalene A	GSM8969898		source name:whole animal\|tissue:whole animal\|genotype:5D WT\|treatment:1 8 dimethylnaphthalene\|batch:1\|geo loc name:missing\|collection date:missing	1 8 dimethylnaphthalene A	Demultiplexing and alignment was performed using the manufacture provided processing platform TempO SeqR BioSpyder Technologies Inc. Carlsbad CA United States. Counts of genes with multiple probes were summed as instructed by the manufacturer resulting in 3112 unique genes Read counts were normalized by upper quartile normalization using the calcNormFactors function from the Bioconductor package in EdgeR using the 75th percentile of reads per samples as the scaling factor. Principal component analysis PCA of normalized read counts was used to determine that a batch effect was present based on exposure and RNA extraction day nested by experimental design. At this time one control sample control day1 B was identified as an outlier and removed from all future analysis. The batch effect was removed from unnormalized data using the ComBat seq function from the SVA R package Normalzied read counts were normalized by upper quartile normalization using the calcNormFactors function from the Bioconductor package in EdgeR using the 75th percentile of reads per samples as the scaling factor. Assembly: GRCz11 Supplementary files format and content: .csv file containing raw count data Supplementary files format and content: .csv file containing batch corrected and normalized data	whole animal	When the embryos were 6hpf they were bleached in groups of 1000 or less. Embryos were placed in a strainer for transfer between solutions in 500 mL containers. First embryos were placed in system fish water for 1 minute then transferred to a 0.5% sodium chloride solution for 5 minutes then transferred to a % sodium hypochlorite solution for 1 minute and rinsed in EM for 5 minutes and transferred to a clean glass petri dish. Embryos were then loaded into 2mL glass exposure vials in groups of 8 using a glass pasture pipette. Excess EM was removed and 2mL of fresh EM was immediately added. Chemical stocks were diluted to 200 times the desired exposure concentration of 20 µM using DMSO. 10 µL of diluted chemical stock was added to each vial vials were immediately capped and inverted 3 times to ensure mixing no precipitate was observed for any chemicals at the concentrations tested. Vials were loaded into a custom 3D printed vial holder for slow end over end rotation in a dark 28°C incubator overnight. At 24 hours embryos were removed from exposure vials and plated into 96 well plates loaded with 100 µL clean EM. At 48 hpf embryos were collected for RNA extraction embryos from two exposure vials were kept as holdback for morphological assessment. From the remaining wells 6 phenotypically normal embryos were collected and pooled in their vial groups into 1.5 mL Eppendorf tubes placed on ice 30 sec excess media removed placed back on ice 30 sec and 200 µL RNA shield added. Five pooled samples were collected from each treatment.	1mL of 1µm beads and 500µL of lysis buffer were loaded into a round bottom 2mL 96 well plate. 100µL of lysis buffer was added to tube containing pooled embryo samples and 200µL of RNA shield the full contents of the tube were then transferred to the homogenization plate. The plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 275 µL of the supernatant was then transferred to two plates containing 275µL of ethanol. RNA extraction was performed using the KingFisher Apex system. Briefly this process uses Mag beads to collect RNA from the sample post series of washes the RNA is eluted in 50µLof ultra pure water. RNA quality was assessed using the Agilent 4150 TapeStation System. All samples used had RIN scores >9.8. RNA concentration was determined using ThermoFisher Quant iT RNA Reagent and assay kit and ThermoFisher Varioskan ALF multimode plate reader in triplicate samples with high variability between reads were repeated in triplicate. The average of the three quantification reads was used for RNA concentration normalization. Each treatment had 4 5 viable RNA extraction replicates for treatments with 5 viable samples the 4 with the highest concentration and RIN score were used 3 control samples were used from each day group. All 94 samples were normalized to the lowest concentration sample 14.9ng/µL. 1 µL per sample of normalized RNA was used as input for library preparation following manufactures protocol for the 96 sample TempO Seq S1500 + Zebrafish Surrogate Assay panel BioSpyder Technologies Inc. Carlsbad CA United States. Briefly samples were added to the assay plate with an equal volume of 2x enhanced lysis buffer and processed through annealing with detector oligos digestion and ligation. Proper amplification was verified using real time PCR green fluorescence. Library purification was performed using the Macherey Nagel NucleoSpin Gel and PCR Cleanup Kit following adjustments detailed in the manufacturer’s instructions. PCR amplification products were pooled into a s…		tissue:whole animal\|genotype:5D WT\|treatment:1 8 dimethylnaphthalene\|batch:1	GSM8969898	GSM8969898: 1 8 dimethylnaphthalene A; Danio rerio; OTHER	GSM8969898 r1	GSM8969898	1	1mL of 1µm beads and 500µL of lysis buffer were loaded into a round bottom 2mL 96 well plate. 100µL of lysis buffer was added to tube containing pooled embryo samples and 200µL of RNA shield the full contents of the tube were then transferred to the homogenization plate. The plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 275 µL of the supernatant was then transferred to two plates containing 275µL of ethanol. RNA extraction was performed using the KingFisher Apex system. Briefly this process uses Mag beads to collect RNA from the sample post series of washes the RNA is eluted in 50µLof ultra pure water. RNA quality was assessed using the Agilent 4150 TapeStation System. All samples used had RIN scores >9.8. RNA concentration was determined using ThermoFisher Quant iT RNA Reagent and assay kit and ThermoFisher Varioskan ALF multimode plate reader in triplicate samples with high variability between reads were repeated in triplicate. The average of the three quantification reads was used for RNA concentration normalization. Each treatment had 4 5 viable RNA extraction replicates for treatments with 5 viable samples the 4 with the highest concentration and RIN score were used 3 control samples were used from each day group. All 94 samples were normalized to the lowest concentration sample 14.9ng/µL. 1 µL per sample of normalized RNA was used as input for library preparation following manufactures protocol for the 96 sample TempO Seq S1500 + Zebrafish Surrogate Assay panel BioSpyder Technologies Inc. Carlsbad CA United States. Briefly samples were added to the assay plate with an equal volume of 2x enhanced lysis buffer and processed through annealing with detector oligos digestion and ligation. Proper amplification was verified using real time PCR green fluorescence. Library purification was performed using the Macherey Nagel NucleoSpin Gel and PCR Cleanup Kit following adjustments detailed in the manufacturer's instructions. PCR amplification products were pooled into a s…	OTHER	TRANSCRIPTOMIC	other	SINGLE	ILLUMINA	Illumina NovaSeq X	SRP583274			2_A.fastq.gz	fastq	610412472.0	11968872.0	GSM8969898 r1	0:51	A:127080561;C:159924680;G:139627579;T:183641966;N:137686	51				127080561	159924680	139627579	183641966	137686	SRX28685529	SRS24952431	SRA2124446	SARL, Environmental and Molecular Toxicology, Oregon State University	SARL, Environmental and Molecular Toxicology, Oregon State University												B		usable mapping rate	illumina	novaseq_era	unknown	other	unknown	bulk	unknown	unknown	United States	2025-05-06	Multi-stage	Embryo	Whole Organism	All anatomical structures
36054	36054	SRR33444766	SRX28685528	SRS24952432	SRP583274	PRJNA1259243	Structure Dependent Developmental Toxicity of Alkyl Substituted Naphthalenes	GSE296410	Transcriptome Analysis	Naphthalene and its alkyl substituted derivatives are among the most abundant polycyclic aromatic hydrocarbons PAHs in environmental and human exposure studies yet their developmental toxicity and mode of action remain poorly understood due to challenges in testing of semi volatile compounds. This study developed a vial based high through put method to effectively assess the activity of naphthalenes and a set of 24 alkyl substituted naphthalenes. Early life stage zebrafish were exposed to a concentration series of each chemical 0 50 µM in rotating sealed glass vials to minimize volatilization. Benchmark concentration BMC50 values were calculated for morphological endpoints and lowest effect levels were determined for behavioral effects. The data were assessed for evidence of a narcotic mode of action using body burden measurements for select chemicals and logKow modeling. Targeted transcriptomics at a single concentration and timepoint as well as in silico molecular docking were conducted to generate mode of action hypothesis. The vial method enabled detection of highly variable developmental toxicity not previously observed using standard 96 well plate exposures. LogKow and body burden were poor predictors of toxicity suggesting a non narcotic mode of action. Transcriptomic analysis revealed a limited but notable set of differentially expressed genes with evidence for the disruption of glucocorticoid signaling pathways. Molecular docking identified potential protein targets e.g. CYP1A2 NT5E FOLR1 that may mediate observed effects. This study demonstrates the importance of appropriate exposure methods for semi volatile compounds reveals structure dependent toxicity among alkyl substituted naphthalenes and provides a foundation for further mechanistic studies and improved risk assessment of alkyl substituted PAHs. Overall design: Zebrafish were exposed to 25 chemicals naphthalene and 24 alkyl sustituted naphthalenes using a vial exposure method. Embryos were exposed to 20 uM of each chemical in groups of…				control day 2 C	GSM8969935		source name:whole animal\|tissue:whole animal\|genotype:5D WT\|treatment:control day 2\|batch:2\|geo loc name:missing\|collection date:missing	control day 2 C	Demultiplexing and alignment was performed using the manufacture provided processing platform TempO SeqR BioSpyder Technologies Inc. Carlsbad CA United States. Counts of genes with multiple probes were summed as instructed by the manufacturer resulting in 3112 unique genes Read counts were normalized by upper quartile normalization using the calcNormFactors function from the Bioconductor package in EdgeR using the 75th percentile of reads per samples as the scaling factor. Principal component analysis PCA of normalized read counts was used to determine that a batch effect was present based on exposure and RNA extraction day nested by experimental design. At this time one control sample control day1 B was identified as an outlier and removed from all future analysis. The batch effect was removed from unnormalized data using the ComBat seq function from the SVA R package Normalzied read counts were normalized by upper quartile normalization using the calcNormFactors function from the Bioconductor package in EdgeR using the 75th percentile of reads per samples as the scaling factor. Assembly: GRCz11 Supplementary files format and content: .csv file containing raw count data Supplementary files format and content: .csv file containing batch corrected and normalized data	whole animal	When the embryos were 6hpf they were bleached in groups of 1000 or less. Embryos were placed in a strainer for transfer between solutions in 500 mL containers. First embryos were placed in system fish water for 1 minute then transferred to a 0.5% sodium chloride solution for 5 minutes then transferred to a % sodium hypochlorite solution for 1 minute and rinsed in EM for 5 minutes and transferred to a clean glass petri dish. Embryos were then loaded into 2mL glass exposure vials in groups of 8 using a glass pasture pipette. Excess EM was removed and 2mL of fresh EM was immediately added. Chemical stocks were diluted to 200 times the desired exposure concentration of 20 µM using DMSO. 10 µL of diluted chemical stock was added to each vial vials were immediately capped and inverted 3 times to ensure mixing no precipitate was observed for any chemicals at the concentrations tested. Vials were loaded into a custom 3D printed vial holder for slow end over end rotation in a dark 28°C incubator overnight. At 24 hours embryos were removed from exposure vials and plated into 96 well plates loaded with 100 µL clean EM. At 48 hpf embryos were collected for RNA extraction embryos from two exposure vials were kept as holdback for morphological assessment. From the remaining wells 6 phenotypically normal embryos were collected and pooled in their vial groups into 1.5 mL Eppendorf tubes placed on ice 30 sec excess media removed placed back on ice 30 sec and 200 µL RNA shield added. Five pooled samples were collected from each treatment.	1mL of 1µm beads and 500µL of lysis buffer were loaded into a round bottom 2mL 96 well plate. 100µL of lysis buffer was added to tube containing pooled embryo samples and 200µL of RNA shield the full contents of the tube were then transferred to the homogenization plate. The plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 275 µL of the supernatant was then transferred to two plates containing 275µL of ethanol. RNA extraction was performed using the KingFisher Apex system. Briefly this process uses Mag beads to collect RNA from the sample post series of washes the RNA is eluted in 50µLof ultra pure water. RNA quality was assessed using the Agilent 4150 TapeStation System. All samples used had RIN scores >9.8. RNA concentration was determined using ThermoFisher Quant iT RNA Reagent and assay kit and ThermoFisher Varioskan ALF multimode plate reader in triplicate samples with high variability between reads were repeated in triplicate. The average of the three quantification reads was used for RNA concentration normalization. Each treatment had 4 5 viable RNA extraction replicates for treatments with 5 viable samples the 4 with the highest concentration and RIN score were used 3 control samples were used from each day group. All 94 samples were normalized to the lowest concentration sample 14.9ng/µL. 1 µL per sample of normalized RNA was used as input for library preparation following manufactures protocol for the 96 sample TempO Seq S1500 + Zebrafish Surrogate Assay panel BioSpyder Technologies Inc. Carlsbad CA United States. Briefly samples were added to the assay plate with an equal volume of 2x enhanced lysis buffer and processed through annealing with detector oligos digestion and ligation. Proper amplification was verified using real time PCR green fluorescence. Library purification was performed using the Macherey Nagel NucleoSpin Gel and PCR Cleanup Kit following adjustments detailed in the manufacturer’s instructions. PCR amplification products were pooled into a s…		tissue:whole animal\|genotype:5D WT\|treatment:control day 2\|batch:2	GSM8969935	GSM8969935: control day 2 C; Danio rerio; OTHER	GSM8969935 r1	GSM8969935	1	1mL of 1µm beads and 500µL of lysis buffer were loaded into a round bottom 2mL 96 well plate. 100µL of lysis buffer was added to tube containing pooled embryo samples and 200µL of RNA shield the full contents of the tube were then transferred to the homogenization plate. The plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 275 µL of the supernatant was then transferred to two plates containing 275µL of ethanol. RNA extraction was performed using the KingFisher Apex system. Briefly this process uses Mag beads to collect RNA from the sample post series of washes the RNA is eluted in 50µLof ultra pure water. RNA quality was assessed using the Agilent 4150 TapeStation System. All samples used had RIN scores >9.8. RNA concentration was determined using ThermoFisher Quant iT RNA Reagent and assay kit and ThermoFisher Varioskan ALF multimode plate reader in triplicate samples with high variability between reads were repeated in triplicate. The average of the three quantification reads was used for RNA concentration normalization. Each treatment had 4 5 viable RNA extraction replicates for treatments with 5 viable samples the 4 with the highest concentration and RIN score were used 3 control samples were used from each day group. All 94 samples were normalized to the lowest concentration sample 14.9ng/µL. 1 µL per sample of normalized RNA was used as input for library preparation following manufactures protocol for the 96 sample TempO Seq S1500 + Zebrafish Surrogate Assay panel BioSpyder Technologies Inc. Carlsbad CA United States. Briefly samples were added to the assay plate with an equal volume of 2x enhanced lysis buffer and processed through annealing with detector oligos digestion and ligation. Proper amplification was verified using real time PCR green fluorescence. Library purification was performed using the Macherey Nagel NucleoSpin Gel and PCR Cleanup Kit following adjustments detailed in the manufacturer's instructions. PCR amplification products were pooled into a s…	OTHER	TRANSCRIPTOMIC	other	SINGLE	ILLUMINA	Illumina NovaSeq X	SRP583274			C2C.fastq.gz	fastq	741413877.0	14537527.0	GSM8969935 r1	0:51	A:153346994;C:193370788;G:172893997;T:221634171;N:167927	51				153346994	193370788	172893997	221634171	167927	SRX28685528	SRS24952432	SRA2124446	SARL, Environmental and Molecular Toxicology, Oregon State University	SARL, Environmental and Molecular Toxicology, Oregon State University												B		usable mapping rate	illumina	novaseq_era	unknown	other	unknown	bulk	unknown	unknown	United States	2025-05-06	Multi-stage	Embryo	Whole Organism	All anatomical structures
36055	36055	SRR33444767	SRX28685527	SRS24952430	SRP583274	PRJNA1259243	Structure Dependent Developmental Toxicity of Alkyl Substituted Naphthalenes	GSE296410	Transcriptome Analysis	Naphthalene and its alkyl substituted derivatives are among the most abundant polycyclic aromatic hydrocarbons PAHs in environmental and human exposure studies yet their developmental toxicity and mode of action remain poorly understood due to challenges in testing of semi volatile compounds. This study developed a vial based high through put method to effectively assess the activity of naphthalenes and a set of 24 alkyl substituted naphthalenes. Early life stage zebrafish were exposed to a concentration series of each chemical 0 50 µM in rotating sealed glass vials to minimize volatilization. Benchmark concentration BMC50 values were calculated for morphological endpoints and lowest effect levels were determined for behavioral effects. The data were assessed for evidence of a narcotic mode of action using body burden measurements for select chemicals and logKow modeling. Targeted transcriptomics at a single concentration and timepoint as well as in silico molecular docking were conducted to generate mode of action hypothesis. The vial method enabled detection of highly variable developmental toxicity not previously observed using standard 96 well plate exposures. LogKow and body burden were poor predictors of toxicity suggesting a non narcotic mode of action. Transcriptomic analysis revealed a limited but notable set of differentially expressed genes with evidence for the disruption of glucocorticoid signaling pathways. Molecular docking identified potential protein targets e.g. CYP1A2 NT5E FOLR1 that may mediate observed effects. This study demonstrates the importance of appropriate exposure methods for semi volatile compounds reveals structure dependent toxicity among alkyl substituted naphthalenes and provides a foundation for further mechanistic studies and improved risk assessment of alkyl substituted PAHs. Overall design: Zebrafish were exposed to 25 chemicals naphthalene and 24 alkyl sustituted naphthalenes using a vial exposure method. Embryos were exposed to 20 uM of each chemical in groups of…				control day 2 B	GSM8969934		source name:whole animal\|tissue:whole animal\|genotype:5D WT\|treatment:control day 2\|batch:2\|geo loc name:missing\|collection date:missing	control day 2 B	Demultiplexing and alignment was performed using the manufacture provided processing platform TempO SeqR BioSpyder Technologies Inc. Carlsbad CA United States. Counts of genes with multiple probes were summed as instructed by the manufacturer resulting in 3112 unique genes Read counts were normalized by upper quartile normalization using the calcNormFactors function from the Bioconductor package in EdgeR using the 75th percentile of reads per samples as the scaling factor. Principal component analysis PCA of normalized read counts was used to determine that a batch effect was present based on exposure and RNA extraction day nested by experimental design. At this time one control sample control day1 B was identified as an outlier and removed from all future analysis. The batch effect was removed from unnormalized data using the ComBat seq function from the SVA R package Normalzied read counts were normalized by upper quartile normalization using the calcNormFactors function from the Bioconductor package in EdgeR using the 75th percentile of reads per samples as the scaling factor. Assembly: GRCz11 Supplementary files format and content: .csv file containing raw count data Supplementary files format and content: .csv file containing batch corrected and normalized data	whole animal	When the embryos were 6hpf they were bleached in groups of 1000 or less. Embryos were placed in a strainer for transfer between solutions in 500 mL containers. First embryos were placed in system fish water for 1 minute then transferred to a 0.5% sodium chloride solution for 5 minutes then transferred to a % sodium hypochlorite solution for 1 minute and rinsed in EM for 5 minutes and transferred to a clean glass petri dish. Embryos were then loaded into 2mL glass exposure vials in groups of 8 using a glass pasture pipette. Excess EM was removed and 2mL of fresh EM was immediately added. Chemical stocks were diluted to 200 times the desired exposure concentration of 20 µM using DMSO. 10 µL of diluted chemical stock was added to each vial vials were immediately capped and inverted 3 times to ensure mixing no precipitate was observed for any chemicals at the concentrations tested. Vials were loaded into a custom 3D printed vial holder for slow end over end rotation in a dark 28°C incubator overnight. At 24 hours embryos were removed from exposure vials and plated into 96 well plates loaded with 100 µL clean EM. At 48 hpf embryos were collected for RNA extraction embryos from two exposure vials were kept as holdback for morphological assessment. From the remaining wells 6 phenotypically normal embryos were collected and pooled in their vial groups into 1.5 mL Eppendorf tubes placed on ice 30 sec excess media removed placed back on ice 30 sec and 200 µL RNA shield added. Five pooled samples were collected from each treatment.	1mL of 1µm beads and 500µL of lysis buffer were loaded into a round bottom 2mL 96 well plate. 100µL of lysis buffer was added to tube containing pooled embryo samples and 200µL of RNA shield the full contents of the tube were then transferred to the homogenization plate. The plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 275 µL of the supernatant was then transferred to two plates containing 275µL of ethanol. RNA extraction was performed using the KingFisher Apex system. Briefly this process uses Mag beads to collect RNA from the sample post series of washes the RNA is eluted in 50µLof ultra pure water. RNA quality was assessed using the Agilent 4150 TapeStation System. All samples used had RIN scores >9.8. RNA concentration was determined using ThermoFisher Quant iT RNA Reagent and assay kit and ThermoFisher Varioskan ALF multimode plate reader in triplicate samples with high variability between reads were repeated in triplicate. The average of the three quantification reads was used for RNA concentration normalization. Each treatment had 4 5 viable RNA extraction replicates for treatments with 5 viable samples the 4 with the highest concentration and RIN score were used 3 control samples were used from each day group. All 94 samples were normalized to the lowest concentration sample 14.9ng/µL. 1 µL per sample of normalized RNA was used as input for library preparation following manufactures protocol for the 96 sample TempO Seq S1500 + Zebrafish Surrogate Assay panel BioSpyder Technologies Inc. Carlsbad CA United States. Briefly samples were added to the assay plate with an equal volume of 2x enhanced lysis buffer and processed through annealing with detector oligos digestion and ligation. Proper amplification was verified using real time PCR green fluorescence. Library purification was performed using the Macherey Nagel NucleoSpin Gel and PCR Cleanup Kit following adjustments detailed in the manufacturer’s instructions. PCR amplification products were pooled into a s…		tissue:whole animal\|genotype:5D WT\|treatment:control day 2\|batch:2	GSM8969934	GSM8969934: control day 2 B; Danio rerio; OTHER	GSM8969934 r1	GSM8969934	1	1mL of 1µm beads and 500µL of lysis buffer were loaded into a round bottom 2mL 96 well plate. 100µL of lysis buffer was added to tube containing pooled embryo samples and 200µL of RNA shield the full contents of the tube were then transferred to the homogenization plate. The plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 275 µL of the supernatant was then transferred to two plates containing 275µL of ethanol. RNA extraction was performed using the KingFisher Apex system. Briefly this process uses Mag beads to collect RNA from the sample post series of washes the RNA is eluted in 50µLof ultra pure water. RNA quality was assessed using the Agilent 4150 TapeStation System. All samples used had RIN scores >9.8. RNA concentration was determined using ThermoFisher Quant iT RNA Reagent and assay kit and ThermoFisher Varioskan ALF multimode plate reader in triplicate samples with high variability between reads were repeated in triplicate. The average of the three quantification reads was used for RNA concentration normalization. Each treatment had 4 5 viable RNA extraction replicates for treatments with 5 viable samples the 4 with the highest concentration and RIN score were used 3 control samples were used from each day group. All 94 samples were normalized to the lowest concentration sample 14.9ng/µL. 1 µL per sample of normalized RNA was used as input for library preparation following manufactures protocol for the 96 sample TempO Seq S1500 + Zebrafish Surrogate Assay panel BioSpyder Technologies Inc. Carlsbad CA United States. Briefly samples were added to the assay plate with an equal volume of 2x enhanced lysis buffer and processed through annealing with detector oligos digestion and ligation. Proper amplification was verified using real time PCR green fluorescence. Library purification was performed using the Macherey Nagel NucleoSpin Gel and PCR Cleanup Kit following adjustments detailed in the manufacturer's instructions. PCR amplification products were pooled into a s…	OTHER	TRANSCRIPTOMIC	other	SINGLE	ILLUMINA	Illumina NovaSeq X	SRP583274			C2B.fastq.gz	fastq	541591338.0	10619438.0	GSM8969934 r1	0:51	A:110558693;C:142536702;G:127264949;T:161109133;N:121861	51				110558693	142536702	127264949	161109133	121861	SRX28685527	SRS24952430	SRA2124446	SARL, Environmental and Molecular Toxicology, Oregon State University	SARL, Environmental and Molecular Toxicology, Oregon State University												B		usable mapping rate	illumina	novaseq_era	unknown	other	unknown	bulk	unknown	unknown	United States	2025-05-06	Multi-stage	Embryo	Whole Organism	All anatomical structures
36056	36056	SRR33444768	SRX28685526	SRS24952429	SRP583274	PRJNA1259243	Structure Dependent Developmental Toxicity of Alkyl Substituted Naphthalenes	GSE296410	Transcriptome Analysis	Naphthalene and its alkyl substituted derivatives are among the most abundant polycyclic aromatic hydrocarbons PAHs in environmental and human exposure studies yet their developmental toxicity and mode of action remain poorly understood due to challenges in testing of semi volatile compounds. This study developed a vial based high through put method to effectively assess the activity of naphthalenes and a set of 24 alkyl substituted naphthalenes. Early life stage zebrafish were exposed to a concentration series of each chemical 0 50 µM in rotating sealed glass vials to minimize volatilization. Benchmark concentration BMC50 values were calculated for morphological endpoints and lowest effect levels were determined for behavioral effects. The data were assessed for evidence of a narcotic mode of action using body burden measurements for select chemicals and logKow modeling. Targeted transcriptomics at a single concentration and timepoint as well as in silico molecular docking were conducted to generate mode of action hypothesis. The vial method enabled detection of highly variable developmental toxicity not previously observed using standard 96 well plate exposures. LogKow and body burden were poor predictors of toxicity suggesting a non narcotic mode of action. Transcriptomic analysis revealed a limited but notable set of differentially expressed genes with evidence for the disruption of glucocorticoid signaling pathways. Molecular docking identified potential protein targets e.g. CYP1A2 NT5E FOLR1 that may mediate observed effects. This study demonstrates the importance of appropriate exposure methods for semi volatile compounds reveals structure dependent toxicity among alkyl substituted naphthalenes and provides a foundation for further mechanistic studies and improved risk assessment of alkyl substituted PAHs. Overall design: Zebrafish were exposed to 25 chemicals naphthalene and 24 alkyl sustituted naphthalenes using a vial exposure method. Embryos were exposed to 20 uM of each chemical in groups of…				control day 2 A	GSM8969933		source name:whole animal\|tissue:whole animal\|genotype:5D WT\|treatment:control day 2\|batch:2\|geo loc name:missing\|collection date:missing	control day 2 A	Demultiplexing and alignment was performed using the manufacture provided processing platform TempO SeqR BioSpyder Technologies Inc. Carlsbad CA United States. Counts of genes with multiple probes were summed as instructed by the manufacturer resulting in 3112 unique genes Read counts were normalized by upper quartile normalization using the calcNormFactors function from the Bioconductor package in EdgeR using the 75th percentile of reads per samples as the scaling factor. Principal component analysis PCA of normalized read counts was used to determine that a batch effect was present based on exposure and RNA extraction day nested by experimental design. At this time one control sample control day1 B was identified as an outlier and removed from all future analysis. The batch effect was removed from unnormalized data using the ComBat seq function from the SVA R package Normalzied read counts were normalized by upper quartile normalization using the calcNormFactors function from the Bioconductor package in EdgeR using the 75th percentile of reads per samples as the scaling factor. Assembly: GRCz11 Supplementary files format and content: .csv file containing raw count data Supplementary files format and content: .csv file containing batch corrected and normalized data	whole animal	When the embryos were 6hpf they were bleached in groups of 1000 or less. Embryos were placed in a strainer for transfer between solutions in 500 mL containers. First embryos were placed in system fish water for 1 minute then transferred to a 0.5% sodium chloride solution for 5 minutes then transferred to a % sodium hypochlorite solution for 1 minute and rinsed in EM for 5 minutes and transferred to a clean glass petri dish. Embryos were then loaded into 2mL glass exposure vials in groups of 8 using a glass pasture pipette. Excess EM was removed and 2mL of fresh EM was immediately added. Chemical stocks were diluted to 200 times the desired exposure concentration of 20 µM using DMSO. 10 µL of diluted chemical stock was added to each vial vials were immediately capped and inverted 3 times to ensure mixing no precipitate was observed for any chemicals at the concentrations tested. Vials were loaded into a custom 3D printed vial holder for slow end over end rotation in a dark 28°C incubator overnight. At 24 hours embryos were removed from exposure vials and plated into 96 well plates loaded with 100 µL clean EM. At 48 hpf embryos were collected for RNA extraction embryos from two exposure vials were kept as holdback for morphological assessment. From the remaining wells 6 phenotypically normal embryos were collected and pooled in their vial groups into 1.5 mL Eppendorf tubes placed on ice 30 sec excess media removed placed back on ice 30 sec and 200 µL RNA shield added. Five pooled samples were collected from each treatment.	1mL of 1µm beads and 500µL of lysis buffer were loaded into a round bottom 2mL 96 well plate. 100µL of lysis buffer was added to tube containing pooled embryo samples and 200µL of RNA shield the full contents of the tube were then transferred to the homogenization plate. The plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 275 µL of the supernatant was then transferred to two plates containing 275µL of ethanol. RNA extraction was performed using the KingFisher Apex system. Briefly this process uses Mag beads to collect RNA from the sample post series of washes the RNA is eluted in 50µLof ultra pure water. RNA quality was assessed using the Agilent 4150 TapeStation System. All samples used had RIN scores >9.8. RNA concentration was determined using ThermoFisher Quant iT RNA Reagent and assay kit and ThermoFisher Varioskan ALF multimode plate reader in triplicate samples with high variability between reads were repeated in triplicate. The average of the three quantification reads was used for RNA concentration normalization. Each treatment had 4 5 viable RNA extraction replicates for treatments with 5 viable samples the 4 with the highest concentration and RIN score were used 3 control samples were used from each day group. All 94 samples were normalized to the lowest concentration sample 14.9ng/µL. 1 µL per sample of normalized RNA was used as input for library preparation following manufactures protocol for the 96 sample TempO Seq S1500 + Zebrafish Surrogate Assay panel BioSpyder Technologies Inc. Carlsbad CA United States. Briefly samples were added to the assay plate with an equal volume of 2x enhanced lysis buffer and processed through annealing with detector oligos digestion and ligation. Proper amplification was verified using real time PCR green fluorescence. Library purification was performed using the Macherey Nagel NucleoSpin Gel and PCR Cleanup Kit following adjustments detailed in the manufacturer’s instructions. PCR amplification products were pooled into a s…		tissue:whole animal\|genotype:5D WT\|treatment:control day 2\|batch:2	GSM8969933	GSM8969933: control day 2 A; Danio rerio; OTHER	GSM8969933 r1	GSM8969933	1	1mL of 1µm beads and 500µL of lysis buffer were loaded into a round bottom 2mL 96 well plate. 100µL of lysis buffer was added to tube containing pooled embryo samples and 200µL of RNA shield the full contents of the tube were then transferred to the homogenization plate. The plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 275 µL of the supernatant was then transferred to two plates containing 275µL of ethanol. RNA extraction was performed using the KingFisher Apex system. Briefly this process uses Mag beads to collect RNA from the sample post series of washes the RNA is eluted in 50µLof ultra pure water. RNA quality was assessed using the Agilent 4150 TapeStation System. All samples used had RIN scores >9.8. RNA concentration was determined using ThermoFisher Quant iT RNA Reagent and assay kit and ThermoFisher Varioskan ALF multimode plate reader in triplicate samples with high variability between reads were repeated in triplicate. The average of the three quantification reads was used for RNA concentration normalization. Each treatment had 4 5 viable RNA extraction replicates for treatments with 5 viable samples the 4 with the highest concentration and RIN score were used 3 control samples were used from each day group. All 94 samples were normalized to the lowest concentration sample 14.9ng/µL. 1 µL per sample of normalized RNA was used as input for library preparation following manufactures protocol for the 96 sample TempO Seq S1500 + Zebrafish Surrogate Assay panel BioSpyder Technologies Inc. Carlsbad CA United States. Briefly samples were added to the assay plate with an equal volume of 2x enhanced lysis buffer and processed through annealing with detector oligos digestion and ligation. Proper amplification was verified using real time PCR green fluorescence. Library purification was performed using the Macherey Nagel NucleoSpin Gel and PCR Cleanup Kit following adjustments detailed in the manufacturer's instructions. PCR amplification products were pooled into a s…	OTHER	TRANSCRIPTOMIC	other	SINGLE	ILLUMINA	Illumina NovaSeq X	SRP583274			C2A.fastq.gz	fastq	613454112.0	12028512.0	GSM8969933 r1	0:51	A:125133985;C:160879875;G:143614243;T:183687282;N:138727	51				125133985	160879875	143614243	183687282	138727	SRX28685526	SRS24952429	SRA2124446	SARL, Environmental and Molecular Toxicology, Oregon State University	SARL, Environmental and Molecular Toxicology, Oregon State University												B		usable mapping rate	illumina	novaseq_era	unknown	other	unknown	bulk	unknown	unknown	United States	2025-05-06	Multi-stage	Embryo	Whole Organism	All anatomical structures
36057	36057	SRR33444769	SRX28685525	SRS24952428	SRP583274	PRJNA1259243	Structure Dependent Developmental Toxicity of Alkyl Substituted Naphthalenes	GSE296410	Transcriptome Analysis	Naphthalene and its alkyl substituted derivatives are among the most abundant polycyclic aromatic hydrocarbons PAHs in environmental and human exposure studies yet their developmental toxicity and mode of action remain poorly understood due to challenges in testing of semi volatile compounds. This study developed a vial based high through put method to effectively assess the activity of naphthalenes and a set of 24 alkyl substituted naphthalenes. Early life stage zebrafish were exposed to a concentration series of each chemical 0 50 µM in rotating sealed glass vials to minimize volatilization. Benchmark concentration BMC50 values were calculated for morphological endpoints and lowest effect levels were determined for behavioral effects. The data were assessed for evidence of a narcotic mode of action using body burden measurements for select chemicals and logKow modeling. Targeted transcriptomics at a single concentration and timepoint as well as in silico molecular docking were conducted to generate mode of action hypothesis. The vial method enabled detection of highly variable developmental toxicity not previously observed using standard 96 well plate exposures. LogKow and body burden were poor predictors of toxicity suggesting a non narcotic mode of action. Transcriptomic analysis revealed a limited but notable set of differentially expressed genes with evidence for the disruption of glucocorticoid signaling pathways. Molecular docking identified potential protein targets e.g. CYP1A2 NT5E FOLR1 that may mediate observed effects. This study demonstrates the importance of appropriate exposure methods for semi volatile compounds reveals structure dependent toxicity among alkyl substituted naphthalenes and provides a foundation for further mechanistic studies and improved risk assessment of alkyl substituted PAHs. Overall design: Zebrafish were exposed to 25 chemicals naphthalene and 24 alkyl sustituted naphthalenes using a vial exposure method. Embryos were exposed to 20 uM of each chemical in groups of…				control day 1 C	GSM8969932		source name:whole animal\|tissue:whole animal\|genotype:5D WT\|treatment:control day 1\|batch:1\|geo loc name:missing\|collection date:missing	control day 1 C	Demultiplexing and alignment was performed using the manufacture provided processing platform TempO SeqR BioSpyder Technologies Inc. Carlsbad CA United States. Counts of genes with multiple probes were summed as instructed by the manufacturer resulting in 3112 unique genes Read counts were normalized by upper quartile normalization using the calcNormFactors function from the Bioconductor package in EdgeR using the 75th percentile of reads per samples as the scaling factor. Principal component analysis PCA of normalized read counts was used to determine that a batch effect was present based on exposure and RNA extraction day nested by experimental design. At this time one control sample control day1 B was identified as an outlier and removed from all future analysis. The batch effect was removed from unnormalized data using the ComBat seq function from the SVA R package Normalzied read counts were normalized by upper quartile normalization using the calcNormFactors function from the Bioconductor package in EdgeR using the 75th percentile of reads per samples as the scaling factor. Assembly: GRCz11 Supplementary files format and content: .csv file containing raw count data Supplementary files format and content: .csv file containing batch corrected and normalized data	whole animal	When the embryos were 6hpf they were bleached in groups of 1000 or less. Embryos were placed in a strainer for transfer between solutions in 500 mL containers. First embryos were placed in system fish water for 1 minute then transferred to a 0.5% sodium chloride solution for 5 minutes then transferred to a % sodium hypochlorite solution for 1 minute and rinsed in EM for 5 minutes and transferred to a clean glass petri dish. Embryos were then loaded into 2mL glass exposure vials in groups of 8 using a glass pasture pipette. Excess EM was removed and 2mL of fresh EM was immediately added. Chemical stocks were diluted to 200 times the desired exposure concentration of 20 µM using DMSO. 10 µL of diluted chemical stock was added to each vial vials were immediately capped and inverted 3 times to ensure mixing no precipitate was observed for any chemicals at the concentrations tested. Vials were loaded into a custom 3D printed vial holder for slow end over end rotation in a dark 28°C incubator overnight. At 24 hours embryos were removed from exposure vials and plated into 96 well plates loaded with 100 µL clean EM. At 48 hpf embryos were collected for RNA extraction embryos from two exposure vials were kept as holdback for morphological assessment. From the remaining wells 6 phenotypically normal embryos were collected and pooled in their vial groups into 1.5 mL Eppendorf tubes placed on ice 30 sec excess media removed placed back on ice 30 sec and 200 µL RNA shield added. Five pooled samples were collected from each treatment.	1mL of 1µm beads and 500µL of lysis buffer were loaded into a round bottom 2mL 96 well plate. 100µL of lysis buffer was added to tube containing pooled embryo samples and 200µL of RNA shield the full contents of the tube were then transferred to the homogenization plate. The plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 275 µL of the supernatant was then transferred to two plates containing 275µL of ethanol. RNA extraction was performed using the KingFisher Apex system. Briefly this process uses Mag beads to collect RNA from the sample post series of washes the RNA is eluted in 50µLof ultra pure water. RNA quality was assessed using the Agilent 4150 TapeStation System. All samples used had RIN scores >9.8. RNA concentration was determined using ThermoFisher Quant iT RNA Reagent and assay kit and ThermoFisher Varioskan ALF multimode plate reader in triplicate samples with high variability between reads were repeated in triplicate. The average of the three quantification reads was used for RNA concentration normalization. Each treatment had 4 5 viable RNA extraction replicates for treatments with 5 viable samples the 4 with the highest concentration and RIN score were used 3 control samples were used from each day group. All 94 samples were normalized to the lowest concentration sample 14.9ng/µL. 1 µL per sample of normalized RNA was used as input for library preparation following manufactures protocol for the 96 sample TempO Seq S1500 + Zebrafish Surrogate Assay panel BioSpyder Technologies Inc. Carlsbad CA United States. Briefly samples were added to the assay plate with an equal volume of 2x enhanced lysis buffer and processed through annealing with detector oligos digestion and ligation. Proper amplification was verified using real time PCR green fluorescence. Library purification was performed using the Macherey Nagel NucleoSpin Gel and PCR Cleanup Kit following adjustments detailed in the manufacturer’s instructions. PCR amplification products were pooled into a s…		tissue:whole animal\|genotype:5D WT\|treatment:control day 1\|batch:1	GSM8969932	GSM8969932: control day 1 C; Danio rerio; OTHER	GSM8969932 r1	GSM8969932	1	1mL of 1µm beads and 500µL of lysis buffer were loaded into a round bottom 2mL 96 well plate. 100µL of lysis buffer was added to tube containing pooled embryo samples and 200µL of RNA shield the full contents of the tube were then transferred to the homogenization plate. The plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 275 µL of the supernatant was then transferred to two plates containing 275µL of ethanol. RNA extraction was performed using the KingFisher Apex system. Briefly this process uses Mag beads to collect RNA from the sample post series of washes the RNA is eluted in 50µLof ultra pure water. RNA quality was assessed using the Agilent 4150 TapeStation System. All samples used had RIN scores >9.8. RNA concentration was determined using ThermoFisher Quant iT RNA Reagent and assay kit and ThermoFisher Varioskan ALF multimode plate reader in triplicate samples with high variability between reads were repeated in triplicate. The average of the three quantification reads was used for RNA concentration normalization. Each treatment had 4 5 viable RNA extraction replicates for treatments with 5 viable samples the 4 with the highest concentration and RIN score were used 3 control samples were used from each day group. All 94 samples were normalized to the lowest concentration sample 14.9ng/µL. 1 µL per sample of normalized RNA was used as input for library preparation following manufactures protocol for the 96 sample TempO Seq S1500 + Zebrafish Surrogate Assay panel BioSpyder Technologies Inc. Carlsbad CA United States. Briefly samples were added to the assay plate with an equal volume of 2x enhanced lysis buffer and processed through annealing with detector oligos digestion and ligation. Proper amplification was verified using real time PCR green fluorescence. Library purification was performed using the Macherey Nagel NucleoSpin Gel and PCR Cleanup Kit following adjustments detailed in the manufacturer's instructions. PCR amplification products were pooled into a s…	OTHER	TRANSCRIPTOMIC	other	SINGLE	ILLUMINA	Illumina NovaSeq X	SRP583274			C1C.fastq.gz	fastq	474767415.0	9309165.0	GSM8969932 r1	0:51	A:97071261;C:124093451;G:110241992;T:143253948;N:106763	51				97071261	124093451	110241992	143253948	106763	SRX28685525	SRS24952428	SRA2124446	SARL, Environmental and Molecular Toxicology, Oregon State University	SARL, Environmental and Molecular Toxicology, Oregon State University												B		usable mapping rate	illumina	novaseq_era	unknown	other	unknown	bulk	unknown	unknown	United States	2025-05-06	Multi-stage	Embryo	Whole Organism	All anatomical structures
36058	36058	SRR33444770	SRX28685524	SRS24952427	SRP583274	PRJNA1259243	Structure Dependent Developmental Toxicity of Alkyl Substituted Naphthalenes	GSE296410	Transcriptome Analysis	Naphthalene and its alkyl substituted derivatives are among the most abundant polycyclic aromatic hydrocarbons PAHs in environmental and human exposure studies yet their developmental toxicity and mode of action remain poorly understood due to challenges in testing of semi volatile compounds. This study developed a vial based high through put method to effectively assess the activity of naphthalenes and a set of 24 alkyl substituted naphthalenes. Early life stage zebrafish were exposed to a concentration series of each chemical 0 50 µM in rotating sealed glass vials to minimize volatilization. Benchmark concentration BMC50 values were calculated for morphological endpoints and lowest effect levels were determined for behavioral effects. The data were assessed for evidence of a narcotic mode of action using body burden measurements for select chemicals and logKow modeling. Targeted transcriptomics at a single concentration and timepoint as well as in silico molecular docking were conducted to generate mode of action hypothesis. The vial method enabled detection of highly variable developmental toxicity not previously observed using standard 96 well plate exposures. LogKow and body burden were poor predictors of toxicity suggesting a non narcotic mode of action. Transcriptomic analysis revealed a limited but notable set of differentially expressed genes with evidence for the disruption of glucocorticoid signaling pathways. Molecular docking identified potential protein targets e.g. CYP1A2 NT5E FOLR1 that may mediate observed effects. This study demonstrates the importance of appropriate exposure methods for semi volatile compounds reveals structure dependent toxicity among alkyl substituted naphthalenes and provides a foundation for further mechanistic studies and improved risk assessment of alkyl substituted PAHs. Overall design: Zebrafish were exposed to 25 chemicals naphthalene and 24 alkyl sustituted naphthalenes using a vial exposure method. Embryos were exposed to 20 uM of each chemical in groups of…				control day 1 B	GSM8969931		source name:whole animal\|tissue:whole animal\|genotype:5D WT\|treatment:control day 1\|batch:1\|geo loc name:missing\|collection date:missing	control day 1 B	Demultiplexing and alignment was performed using the manufacture provided processing platform TempO SeqR BioSpyder Technologies Inc. Carlsbad CA United States. Counts of genes with multiple probes were summed as instructed by the manufacturer resulting in 3112 unique genes Read counts were normalized by upper quartile normalization using the calcNormFactors function from the Bioconductor package in EdgeR using the 75th percentile of reads per samples as the scaling factor. Principal component analysis PCA of normalized read counts was used to determine that a batch effect was present based on exposure and RNA extraction day nested by experimental design. At this time one control sample control day1 B was identified as an outlier and removed from all future analysis. The batch effect was removed from unnormalized data using the ComBat seq function from the SVA R package Normalzied read counts were normalized by upper quartile normalization using the calcNormFactors function from the Bioconductor package in EdgeR using the 75th percentile of reads per samples as the scaling factor. Assembly: GRCz11 Supplementary files format and content: .csv file containing raw count data Supplementary files format and content: .csv file containing batch corrected and normalized data	whole animal	When the embryos were 6hpf they were bleached in groups of 1000 or less. Embryos were placed in a strainer for transfer between solutions in 500 mL containers. First embryos were placed in system fish water for 1 minute then transferred to a 0.5% sodium chloride solution for 5 minutes then transferred to a % sodium hypochlorite solution for 1 minute and rinsed in EM for 5 minutes and transferred to a clean glass petri dish. Embryos were then loaded into 2mL glass exposure vials in groups of 8 using a glass pasture pipette. Excess EM was removed and 2mL of fresh EM was immediately added. Chemical stocks were diluted to 200 times the desired exposure concentration of 20 µM using DMSO. 10 µL of diluted chemical stock was added to each vial vials were immediately capped and inverted 3 times to ensure mixing no precipitate was observed for any chemicals at the concentrations tested. Vials were loaded into a custom 3D printed vial holder for slow end over end rotation in a dark 28°C incubator overnight. At 24 hours embryos were removed from exposure vials and plated into 96 well plates loaded with 100 µL clean EM. At 48 hpf embryos were collected for RNA extraction embryos from two exposure vials were kept as holdback for morphological assessment. From the remaining wells 6 phenotypically normal embryos were collected and pooled in their vial groups into 1.5 mL Eppendorf tubes placed on ice 30 sec excess media removed placed back on ice 30 sec and 200 µL RNA shield added. Five pooled samples were collected from each treatment.	1mL of 1µm beads and 500µL of lysis buffer were loaded into a round bottom 2mL 96 well plate. 100µL of lysis buffer was added to tube containing pooled embryo samples and 200µL of RNA shield the full contents of the tube were then transferred to the homogenization plate. The plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 275 µL of the supernatant was then transferred to two plates containing 275µL of ethanol. RNA extraction was performed using the KingFisher Apex system. Briefly this process uses Mag beads to collect RNA from the sample post series of washes the RNA is eluted in 50µLof ultra pure water. RNA quality was assessed using the Agilent 4150 TapeStation System. All samples used had RIN scores >9.8. RNA concentration was determined using ThermoFisher Quant iT RNA Reagent and assay kit and ThermoFisher Varioskan ALF multimode plate reader in triplicate samples with high variability between reads were repeated in triplicate. The average of the three quantification reads was used for RNA concentration normalization. Each treatment had 4 5 viable RNA extraction replicates for treatments with 5 viable samples the 4 with the highest concentration and RIN score were used 3 control samples were used from each day group. All 94 samples were normalized to the lowest concentration sample 14.9ng/µL. 1 µL per sample of normalized RNA was used as input for library preparation following manufactures protocol for the 96 sample TempO Seq S1500 + Zebrafish Surrogate Assay panel BioSpyder Technologies Inc. Carlsbad CA United States. Briefly samples were added to the assay plate with an equal volume of 2x enhanced lysis buffer and processed through annealing with detector oligos digestion and ligation. Proper amplification was verified using real time PCR green fluorescence. Library purification was performed using the Macherey Nagel NucleoSpin Gel and PCR Cleanup Kit following adjustments detailed in the manufacturer’s instructions. PCR amplification products were pooled into a s…		tissue:whole animal\|genotype:5D WT\|treatment:control day 1\|batch:1	GSM8969931	GSM8969931: control day 1 B; Danio rerio; OTHER	GSM8969931 r1	GSM8969931	1	1mL of 1µm beads and 500µL of lysis buffer were loaded into a round bottom 2mL 96 well plate. 100µL of lysis buffer was added to tube containing pooled embryo samples and 200µL of RNA shield the full contents of the tube were then transferred to the homogenization plate. The plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 275 µL of the supernatant was then transferred to two plates containing 275µL of ethanol. RNA extraction was performed using the KingFisher Apex system. Briefly this process uses Mag beads to collect RNA from the sample post series of washes the RNA is eluted in 50µLof ultra pure water. RNA quality was assessed using the Agilent 4150 TapeStation System. All samples used had RIN scores >9.8. RNA concentration was determined using ThermoFisher Quant iT RNA Reagent and assay kit and ThermoFisher Varioskan ALF multimode plate reader in triplicate samples with high variability between reads were repeated in triplicate. The average of the three quantification reads was used for RNA concentration normalization. Each treatment had 4 5 viable RNA extraction replicates for treatments with 5 viable samples the 4 with the highest concentration and RIN score were used 3 control samples were used from each day group. All 94 samples were normalized to the lowest concentration sample 14.9ng/µL. 1 µL per sample of normalized RNA was used as input for library preparation following manufactures protocol for the 96 sample TempO Seq S1500 + Zebrafish Surrogate Assay panel BioSpyder Technologies Inc. Carlsbad CA United States. Briefly samples were added to the assay plate with an equal volume of 2x enhanced lysis buffer and processed through annealing with detector oligos digestion and ligation. Proper amplification was verified using real time PCR green fluorescence. Library purification was performed using the Macherey Nagel NucleoSpin Gel and PCR Cleanup Kit following adjustments detailed in the manufacturer's instructions. PCR amplification products were pooled into a s…	OTHER	TRANSCRIPTOMIC	other	SINGLE	ILLUMINA	Illumina NovaSeq X	SRP583274			C1B.fastq.gz	fastq	457427772.0	8969172.0	GSM8969931 r1	0:51	A:94621194;C:123105730;G:103779476;T:135818957;N:102415	51				94621194	123105730	103779476	135818957	102415	SRX28685524	SRS24952427	SRA2124446	SARL, Environmental and Molecular Toxicology, Oregon State University	SARL, Environmental and Molecular Toxicology, Oregon State University												B		usable mapping rate	illumina	novaseq_era	unknown	other	unknown	bulk	unknown	unknown	United States	2025-05-06	Multi-stage	Embryo	Whole Organism	All anatomical structures
36059	36059	SRR33444771	SRX28685523	SRS24952426	SRP583274	PRJNA1259243	Structure Dependent Developmental Toxicity of Alkyl Substituted Naphthalenes	GSE296410	Transcriptome Analysis	Naphthalene and its alkyl substituted derivatives are among the most abundant polycyclic aromatic hydrocarbons PAHs in environmental and human exposure studies yet their developmental toxicity and mode of action remain poorly understood due to challenges in testing of semi volatile compounds. This study developed a vial based high through put method to effectively assess the activity of naphthalenes and a set of 24 alkyl substituted naphthalenes. Early life stage zebrafish were exposed to a concentration series of each chemical 0 50 µM in rotating sealed glass vials to minimize volatilization. Benchmark concentration BMC50 values were calculated for morphological endpoints and lowest effect levels were determined for behavioral effects. The data were assessed for evidence of a narcotic mode of action using body burden measurements for select chemicals and logKow modeling. Targeted transcriptomics at a single concentration and timepoint as well as in silico molecular docking were conducted to generate mode of action hypothesis. The vial method enabled detection of highly variable developmental toxicity not previously observed using standard 96 well plate exposures. LogKow and body burden were poor predictors of toxicity suggesting a non narcotic mode of action. Transcriptomic analysis revealed a limited but notable set of differentially expressed genes with evidence for the disruption of glucocorticoid signaling pathways. Molecular docking identified potential protein targets e.g. CYP1A2 NT5E FOLR1 that may mediate observed effects. This study demonstrates the importance of appropriate exposure methods for semi volatile compounds reveals structure dependent toxicity among alkyl substituted naphthalenes and provides a foundation for further mechanistic studies and improved risk assessment of alkyl substituted PAHs. Overall design: Zebrafish were exposed to 25 chemicals naphthalene and 24 alkyl sustituted naphthalenes using a vial exposure method. Embryos were exposed to 20 uM of each chemical in groups of…				control day 1 A	GSM8969930		source name:whole animal\|tissue:whole animal\|genotype:5D WT\|treatment:control day 1\|batch:1\|geo loc name:missing\|collection date:missing	control day 1 A	Demultiplexing and alignment was performed using the manufacture provided processing platform TempO SeqR BioSpyder Technologies Inc. Carlsbad CA United States. Counts of genes with multiple probes were summed as instructed by the manufacturer resulting in 3112 unique genes Read counts were normalized by upper quartile normalization using the calcNormFactors function from the Bioconductor package in EdgeR using the 75th percentile of reads per samples as the scaling factor. Principal component analysis PCA of normalized read counts was used to determine that a batch effect was present based on exposure and RNA extraction day nested by experimental design. At this time one control sample control day1 B was identified as an outlier and removed from all future analysis. The batch effect was removed from unnormalized data using the ComBat seq function from the SVA R package Normalzied read counts were normalized by upper quartile normalization using the calcNormFactors function from the Bioconductor package in EdgeR using the 75th percentile of reads per samples as the scaling factor. Assembly: GRCz11 Supplementary files format and content: .csv file containing raw count data Supplementary files format and content: .csv file containing batch corrected and normalized data	whole animal	When the embryos were 6hpf they were bleached in groups of 1000 or less. Embryos were placed in a strainer for transfer between solutions in 500 mL containers. First embryos were placed in system fish water for 1 minute then transferred to a 0.5% sodium chloride solution for 5 minutes then transferred to a % sodium hypochlorite solution for 1 minute and rinsed in EM for 5 minutes and transferred to a clean glass petri dish. Embryos were then loaded into 2mL glass exposure vials in groups of 8 using a glass pasture pipette. Excess EM was removed and 2mL of fresh EM was immediately added. Chemical stocks were diluted to 200 times the desired exposure concentration of 20 µM using DMSO. 10 µL of diluted chemical stock was added to each vial vials were immediately capped and inverted 3 times to ensure mixing no precipitate was observed for any chemicals at the concentrations tested. Vials were loaded into a custom 3D printed vial holder for slow end over end rotation in a dark 28°C incubator overnight. At 24 hours embryos were removed from exposure vials and plated into 96 well plates loaded with 100 µL clean EM. At 48 hpf embryos were collected for RNA extraction embryos from two exposure vials were kept as holdback for morphological assessment. From the remaining wells 6 phenotypically normal embryos were collected and pooled in their vial groups into 1.5 mL Eppendorf tubes placed on ice 30 sec excess media removed placed back on ice 30 sec and 200 µL RNA shield added. Five pooled samples were collected from each treatment.	1mL of 1µm beads and 500µL of lysis buffer were loaded into a round bottom 2mL 96 well plate. 100µL of lysis buffer was added to tube containing pooled embryo samples and 200µL of RNA shield the full contents of the tube were then transferred to the homogenization plate. The plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 275 µL of the supernatant was then transferred to two plates containing 275µL of ethanol. RNA extraction was performed using the KingFisher Apex system. Briefly this process uses Mag beads to collect RNA from the sample post series of washes the RNA is eluted in 50µLof ultra pure water. RNA quality was assessed using the Agilent 4150 TapeStation System. All samples used had RIN scores >9.8. RNA concentration was determined using ThermoFisher Quant iT RNA Reagent and assay kit and ThermoFisher Varioskan ALF multimode plate reader in triplicate samples with high variability between reads were repeated in triplicate. The average of the three quantification reads was used for RNA concentration normalization. Each treatment had 4 5 viable RNA extraction replicates for treatments with 5 viable samples the 4 with the highest concentration and RIN score were used 3 control samples were used from each day group. All 94 samples were normalized to the lowest concentration sample 14.9ng/µL. 1 µL per sample of normalized RNA was used as input for library preparation following manufactures protocol for the 96 sample TempO Seq S1500 + Zebrafish Surrogate Assay panel BioSpyder Technologies Inc. Carlsbad CA United States. Briefly samples were added to the assay plate with an equal volume of 2x enhanced lysis buffer and processed through annealing with detector oligos digestion and ligation. Proper amplification was verified using real time PCR green fluorescence. Library purification was performed using the Macherey Nagel NucleoSpin Gel and PCR Cleanup Kit following adjustments detailed in the manufacturer’s instructions. PCR amplification products were pooled into a s…		tissue:whole animal\|genotype:5D WT\|treatment:control day 1\|batch:1	GSM8969930	GSM8969930: control day 1 A; Danio rerio; OTHER	GSM8969930 r1	GSM8969930	1	1mL of 1µm beads and 500µL of lysis buffer were loaded into a round bottom 2mL 96 well plate. 100µL of lysis buffer was added to tube containing pooled embryo samples and 200µL of RNA shield the full contents of the tube were then transferred to the homogenization plate. The plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 275 µL of the supernatant was then transferred to two plates containing 275µL of ethanol. RNA extraction was performed using the KingFisher Apex system. Briefly this process uses Mag beads to collect RNA from the sample post series of washes the RNA is eluted in 50µLof ultra pure water. RNA quality was assessed using the Agilent 4150 TapeStation System. All samples used had RIN scores >9.8. RNA concentration was determined using ThermoFisher Quant iT RNA Reagent and assay kit and ThermoFisher Varioskan ALF multimode plate reader in triplicate samples with high variability between reads were repeated in triplicate. The average of the three quantification reads was used for RNA concentration normalization. Each treatment had 4 5 viable RNA extraction replicates for treatments with 5 viable samples the 4 with the highest concentration and RIN score were used 3 control samples were used from each day group. All 94 samples were normalized to the lowest concentration sample 14.9ng/µL. 1 µL per sample of normalized RNA was used as input for library preparation following manufactures protocol for the 96 sample TempO Seq S1500 + Zebrafish Surrogate Assay panel BioSpyder Technologies Inc. Carlsbad CA United States. Briefly samples were added to the assay plate with an equal volume of 2x enhanced lysis buffer and processed through annealing with detector oligos digestion and ligation. Proper amplification was verified using real time PCR green fluorescence. Library purification was performed using the Macherey Nagel NucleoSpin Gel and PCR Cleanup Kit following adjustments detailed in the manufacturer's instructions. PCR amplification products were pooled into a s…	OTHER	TRANSCRIPTOMIC	other	SINGLE	ILLUMINA	Illumina NovaSeq X	SRP583274			C1A.fastq.gz	fastq	492735021.0	9661471.0	GSM8969930 r1	0:51	A:101186692;C:127371147;G:115048213;T:149017307;N:111662	51				101186692	127371147	115048213	149017307	111662	SRX28685523	SRS24952426	SRA2124446	SARL, Environmental and Molecular Toxicology, Oregon State University	SARL, Environmental and Molecular Toxicology, Oregon State University												B		usable mapping rate	illumina	novaseq_era	unknown	other	unknown	bulk	unknown	unknown	United States	2025-05-06	Multi-stage	Embryo	Whole Organism	All anatomical structures
36060	36060	SRR33444772	SRX28685522	SRS24952423	SRP583274	PRJNA1259243	Structure Dependent Developmental Toxicity of Alkyl Substituted Naphthalenes	GSE296410	Transcriptome Analysis	Naphthalene and its alkyl substituted derivatives are among the most abundant polycyclic aromatic hydrocarbons PAHs in environmental and human exposure studies yet their developmental toxicity and mode of action remain poorly understood due to challenges in testing of semi volatile compounds. This study developed a vial based high through put method to effectively assess the activity of naphthalenes and a set of 24 alkyl substituted naphthalenes. Early life stage zebrafish were exposed to a concentration series of each chemical 0 50 µM in rotating sealed glass vials to minimize volatilization. Benchmark concentration BMC50 values were calculated for morphological endpoints and lowest effect levels were determined for behavioral effects. The data were assessed for evidence of a narcotic mode of action using body burden measurements for select chemicals and logKow modeling. Targeted transcriptomics at a single concentration and timepoint as well as in silico molecular docking were conducted to generate mode of action hypothesis. The vial method enabled detection of highly variable developmental toxicity not previously observed using standard 96 well plate exposures. LogKow and body burden were poor predictors of toxicity suggesting a non narcotic mode of action. Transcriptomic analysis revealed a limited but notable set of differentially expressed genes with evidence for the disruption of glucocorticoid signaling pathways. Molecular docking identified potential protein targets e.g. CYP1A2 NT5E FOLR1 that may mediate observed effects. This study demonstrates the importance of appropriate exposure methods for semi volatile compounds reveals structure dependent toxicity among alkyl substituted naphthalenes and provides a foundation for further mechanistic studies and improved risk assessment of alkyl substituted PAHs. Overall design: Zebrafish were exposed to 25 chemicals naphthalene and 24 alkyl sustituted naphthalenes using a vial exposure method. Embryos were exposed to 20 uM of each chemical in groups of…				1 6 7 trimethylnaphthalene E	GSM8969929		source name:whole animal\|tissue:whole animal\|genotype:5D WT\|treatment:1 6 7 trimethylnaphthalene\|batch:1\|geo loc name:missing\|collection date:missing	1 6 7 trimethylnaphthalene E	Demultiplexing and alignment was performed using the manufacture provided processing platform TempO SeqR BioSpyder Technologies Inc. Carlsbad CA United States. Counts of genes with multiple probes were summed as instructed by the manufacturer resulting in 3112 unique genes Read counts were normalized by upper quartile normalization using the calcNormFactors function from the Bioconductor package in EdgeR using the 75th percentile of reads per samples as the scaling factor. Principal component analysis PCA of normalized read counts was used to determine that a batch effect was present based on exposure and RNA extraction day nested by experimental design. At this time one control sample control day1 B was identified as an outlier and removed from all future analysis. The batch effect was removed from unnormalized data using the ComBat seq function from the SVA R package Normalzied read counts were normalized by upper quartile normalization using the calcNormFactors function from the Bioconductor package in EdgeR using the 75th percentile of reads per samples as the scaling factor. Assembly: GRCz11 Supplementary files format and content: .csv file containing raw count data Supplementary files format and content: .csv file containing batch corrected and normalized data	whole animal	When the embryos were 6hpf they were bleached in groups of 1000 or less. Embryos were placed in a strainer for transfer between solutions in 500 mL containers. First embryos were placed in system fish water for 1 minute then transferred to a 0.5% sodium chloride solution for 5 minutes then transferred to a % sodium hypochlorite solution for 1 minute and rinsed in EM for 5 minutes and transferred to a clean glass petri dish. Embryos were then loaded into 2mL glass exposure vials in groups of 8 using a glass pasture pipette. Excess EM was removed and 2mL of fresh EM was immediately added. Chemical stocks were diluted to 200 times the desired exposure concentration of 20 µM using DMSO. 10 µL of diluted chemical stock was added to each vial vials were immediately capped and inverted 3 times to ensure mixing no precipitate was observed for any chemicals at the concentrations tested. Vials were loaded into a custom 3D printed vial holder for slow end over end rotation in a dark 28°C incubator overnight. At 24 hours embryos were removed from exposure vials and plated into 96 well plates loaded with 100 µL clean EM. At 48 hpf embryos were collected for RNA extraction embryos from two exposure vials were kept as holdback for morphological assessment. From the remaining wells 6 phenotypically normal embryos were collected and pooled in their vial groups into 1.5 mL Eppendorf tubes placed on ice 30 sec excess media removed placed back on ice 30 sec and 200 µL RNA shield added. Five pooled samples were collected from each treatment.	1mL of 1µm beads and 500µL of lysis buffer were loaded into a round bottom 2mL 96 well plate. 100µL of lysis buffer was added to tube containing pooled embryo samples and 200µL of RNA shield the full contents of the tube were then transferred to the homogenization plate. The plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 275 µL of the supernatant was then transferred to two plates containing 275µL of ethanol. RNA extraction was performed using the KingFisher Apex system. Briefly this process uses Mag beads to collect RNA from the sample post series of washes the RNA is eluted in 50µLof ultra pure water. RNA quality was assessed using the Agilent 4150 TapeStation System. All samples used had RIN scores >9.8. RNA concentration was determined using ThermoFisher Quant iT RNA Reagent and assay kit and ThermoFisher Varioskan ALF multimode plate reader in triplicate samples with high variability between reads were repeated in triplicate. The average of the three quantification reads was used for RNA concentration normalization. Each treatment had 4 5 viable RNA extraction replicates for treatments with 5 viable samples the 4 with the highest concentration and RIN score were used 3 control samples were used from each day group. All 94 samples were normalized to the lowest concentration sample 14.9ng/µL. 1 µL per sample of normalized RNA was used as input for library preparation following manufactures protocol for the 96 sample TempO Seq S1500 + Zebrafish Surrogate Assay panel BioSpyder Technologies Inc. Carlsbad CA United States. Briefly samples were added to the assay plate with an equal volume of 2x enhanced lysis buffer and processed through annealing with detector oligos digestion and ligation. Proper amplification was verified using real time PCR green fluorescence. Library purification was performed using the Macherey Nagel NucleoSpin Gel and PCR Cleanup Kit following adjustments detailed in the manufacturer’s instructions. PCR amplification products were pooled into a s…		tissue:whole animal\|genotype:5D WT\|treatment:1 6 7 trimethylnaphthalene\|batch:1	GSM8969929	GSM8969929: 1 6 7 trimethylnaphthalene E; Danio rerio; OTHER	GSM8969929 r1	GSM8969929	1	1mL of 1µm beads and 500µL of lysis buffer were loaded into a round bottom 2mL 96 well plate. 100µL of lysis buffer was added to tube containing pooled embryo samples and 200µL of RNA shield the full contents of the tube were then transferred to the homogenization plate. The plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 275 µL of the supernatant was then transferred to two plates containing 275µL of ethanol. RNA extraction was performed using the KingFisher Apex system. Briefly this process uses Mag beads to collect RNA from the sample post series of washes the RNA is eluted in 50µLof ultra pure water. RNA quality was assessed using the Agilent 4150 TapeStation System. All samples used had RIN scores >9.8. RNA concentration was determined using ThermoFisher Quant iT RNA Reagent and assay kit and ThermoFisher Varioskan ALF multimode plate reader in triplicate samples with high variability between reads were repeated in triplicate. The average of the three quantification reads was used for RNA concentration normalization. Each treatment had 4 5 viable RNA extraction replicates for treatments with 5 viable samples the 4 with the highest concentration and RIN score were used 3 control samples were used from each day group. All 94 samples were normalized to the lowest concentration sample 14.9ng/µL. 1 µL per sample of normalized RNA was used as input for library preparation following manufactures protocol for the 96 sample TempO Seq S1500 + Zebrafish Surrogate Assay panel BioSpyder Technologies Inc. Carlsbad CA United States. Briefly samples were added to the assay plate with an equal volume of 2x enhanced lysis buffer and processed through annealing with detector oligos digestion and ligation. Proper amplification was verified using real time PCR green fluorescence. Library purification was performed using the Macherey Nagel NucleoSpin Gel and PCR Cleanup Kit following adjustments detailed in the manufacturer's instructions. PCR amplification products were pooled into a s…	OTHER	TRANSCRIPTOMIC	other	SINGLE	ILLUMINA	Illumina NovaSeq X	SRP583274			9_E.fastq.gz	fastq	611528913.0	11990763.0	GSM8969929 r1	0:51	A:124350552;C:160747373;G:139974969;T:186316449;N:139570	51				124350552	160747373	139974969	186316449	139570	SRX28685522	SRS24952423	SRA2124446	SARL, Environmental and Molecular Toxicology, Oregon State University	SARL, Environmental and Molecular Toxicology, Oregon State University												B		usable mapping rate	illumina	novaseq_era	unknown	other	unknown	bulk	unknown	unknown	United States	2025-05-06	Multi-stage	Embryo	Whole Organism	All anatomical structures
36061	36061	SRR33444773	SRX28685521	SRS24952425	SRP583274	PRJNA1259243	Structure Dependent Developmental Toxicity of Alkyl Substituted Naphthalenes	GSE296410	Transcriptome Analysis	Naphthalene and its alkyl substituted derivatives are among the most abundant polycyclic aromatic hydrocarbons PAHs in environmental and human exposure studies yet their developmental toxicity and mode of action remain poorly understood due to challenges in testing of semi volatile compounds. This study developed a vial based high through put method to effectively assess the activity of naphthalenes and a set of 24 alkyl substituted naphthalenes. Early life stage zebrafish were exposed to a concentration series of each chemical 0 50 µM in rotating sealed glass vials to minimize volatilization. Benchmark concentration BMC50 values were calculated for morphological endpoints and lowest effect levels were determined for behavioral effects. The data were assessed for evidence of a narcotic mode of action using body burden measurements for select chemicals and logKow modeling. Targeted transcriptomics at a single concentration and timepoint as well as in silico molecular docking were conducted to generate mode of action hypothesis. The vial method enabled detection of highly variable developmental toxicity not previously observed using standard 96 well plate exposures. LogKow and body burden were poor predictors of toxicity suggesting a non narcotic mode of action. Transcriptomic analysis revealed a limited but notable set of differentially expressed genes with evidence for the disruption of glucocorticoid signaling pathways. Molecular docking identified potential protein targets e.g. CYP1A2 NT5E FOLR1 that may mediate observed effects. This study demonstrates the importance of appropriate exposure methods for semi volatile compounds reveals structure dependent toxicity among alkyl substituted naphthalenes and provides a foundation for further mechanistic studies and improved risk assessment of alkyl substituted PAHs. Overall design: Zebrafish were exposed to 25 chemicals naphthalene and 24 alkyl sustituted naphthalenes using a vial exposure method. Embryos were exposed to 20 uM of each chemical in groups of…				1 6 7 trimethylnaphthalene D	GSM8969928		source name:whole animal\|tissue:whole animal\|genotype:5D WT\|treatment:1 6 7 trimethylnaphthalene\|batch:1\|geo loc name:missing\|collection date:missing	1 6 7 trimethylnaphthalene D	Demultiplexing and alignment was performed using the manufacture provided processing platform TempO SeqR BioSpyder Technologies Inc. Carlsbad CA United States. Counts of genes with multiple probes were summed as instructed by the manufacturer resulting in 3112 unique genes Read counts were normalized by upper quartile normalization using the calcNormFactors function from the Bioconductor package in EdgeR using the 75th percentile of reads per samples as the scaling factor. Principal component analysis PCA of normalized read counts was used to determine that a batch effect was present based on exposure and RNA extraction day nested by experimental design. At this time one control sample control day1 B was identified as an outlier and removed from all future analysis. The batch effect was removed from unnormalized data using the ComBat seq function from the SVA R package Normalzied read counts were normalized by upper quartile normalization using the calcNormFactors function from the Bioconductor package in EdgeR using the 75th percentile of reads per samples as the scaling factor. Assembly: GRCz11 Supplementary files format and content: .csv file containing raw count data Supplementary files format and content: .csv file containing batch corrected and normalized data	whole animal	When the embryos were 6hpf they were bleached in groups of 1000 or less. Embryos were placed in a strainer for transfer between solutions in 500 mL containers. First embryos were placed in system fish water for 1 minute then transferred to a 0.5% sodium chloride solution for 5 minutes then transferred to a % sodium hypochlorite solution for 1 minute and rinsed in EM for 5 minutes and transferred to a clean glass petri dish. Embryos were then loaded into 2mL glass exposure vials in groups of 8 using a glass pasture pipette. Excess EM was removed and 2mL of fresh EM was immediately added. Chemical stocks were diluted to 200 times the desired exposure concentration of 20 µM using DMSO. 10 µL of diluted chemical stock was added to each vial vials were immediately capped and inverted 3 times to ensure mixing no precipitate was observed for any chemicals at the concentrations tested. Vials were loaded into a custom 3D printed vial holder for slow end over end rotation in a dark 28°C incubator overnight. At 24 hours embryos were removed from exposure vials and plated into 96 well plates loaded with 100 µL clean EM. At 48 hpf embryos were collected for RNA extraction embryos from two exposure vials were kept as holdback for morphological assessment. From the remaining wells 6 phenotypically normal embryos were collected and pooled in their vial groups into 1.5 mL Eppendorf tubes placed on ice 30 sec excess media removed placed back on ice 30 sec and 200 µL RNA shield added. Five pooled samples were collected from each treatment.	1mL of 1µm beads and 500µL of lysis buffer were loaded into a round bottom 2mL 96 well plate. 100µL of lysis buffer was added to tube containing pooled embryo samples and 200µL of RNA shield the full contents of the tube were then transferred to the homogenization plate. The plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 275 µL of the supernatant was then transferred to two plates containing 275µL of ethanol. RNA extraction was performed using the KingFisher Apex system. Briefly this process uses Mag beads to collect RNA from the sample post series of washes the RNA is eluted in 50µLof ultra pure water. RNA quality was assessed using the Agilent 4150 TapeStation System. All samples used had RIN scores >9.8. RNA concentration was determined using ThermoFisher Quant iT RNA Reagent and assay kit and ThermoFisher Varioskan ALF multimode plate reader in triplicate samples with high variability between reads were repeated in triplicate. The average of the three quantification reads was used for RNA concentration normalization. Each treatment had 4 5 viable RNA extraction replicates for treatments with 5 viable samples the 4 with the highest concentration and RIN score were used 3 control samples were used from each day group. All 94 samples were normalized to the lowest concentration sample 14.9ng/µL. 1 µL per sample of normalized RNA was used as input for library preparation following manufactures protocol for the 96 sample TempO Seq S1500 + Zebrafish Surrogate Assay panel BioSpyder Technologies Inc. Carlsbad CA United States. Briefly samples were added to the assay plate with an equal volume of 2x enhanced lysis buffer and processed through annealing with detector oligos digestion and ligation. Proper amplification was verified using real time PCR green fluorescence. Library purification was performed using the Macherey Nagel NucleoSpin Gel and PCR Cleanup Kit following adjustments detailed in the manufacturer’s instructions. PCR amplification products were pooled into a s…		tissue:whole animal\|genotype:5D WT\|treatment:1 6 7 trimethylnaphthalene\|batch:1	GSM8969928	GSM8969928: 1 6 7 trimethylnaphthalene D; Danio rerio; OTHER	GSM8969928 r1	GSM8969928	1	1mL of 1µm beads and 500µL of lysis buffer were loaded into a round bottom 2mL 96 well plate. 100µL of lysis buffer was added to tube containing pooled embryo samples and 200µL of RNA shield the full contents of the tube were then transferred to the homogenization plate. The plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 275 µL of the supernatant was then transferred to two plates containing 275µL of ethanol. RNA extraction was performed using the KingFisher Apex system. Briefly this process uses Mag beads to collect RNA from the sample post series of washes the RNA is eluted in 50µLof ultra pure water. RNA quality was assessed using the Agilent 4150 TapeStation System. All samples used had RIN scores >9.8. RNA concentration was determined using ThermoFisher Quant iT RNA Reagent and assay kit and ThermoFisher Varioskan ALF multimode plate reader in triplicate samples with high variability between reads were repeated in triplicate. The average of the three quantification reads was used for RNA concentration normalization. Each treatment had 4 5 viable RNA extraction replicates for treatments with 5 viable samples the 4 with the highest concentration and RIN score were used 3 control samples were used from each day group. All 94 samples were normalized to the lowest concentration sample 14.9ng/µL. 1 µL per sample of normalized RNA was used as input for library preparation following manufactures protocol for the 96 sample TempO Seq S1500 + Zebrafish Surrogate Assay panel BioSpyder Technologies Inc. Carlsbad CA United States. Briefly samples were added to the assay plate with an equal volume of 2x enhanced lysis buffer and processed through annealing with detector oligos digestion and ligation. Proper amplification was verified using real time PCR green fluorescence. Library purification was performed using the Macherey Nagel NucleoSpin Gel and PCR Cleanup Kit following adjustments detailed in the manufacturer's instructions. PCR amplification products were pooled into a s…	OTHER	TRANSCRIPTOMIC	other	SINGLE	ILLUMINA	Illumina NovaSeq X	SRP583274			9_D.fastq.gz	fastq	628858662.0	12330562.0	GSM8969928 r1	0:51	A:128538964;C:164901356;G:144434115;T:190840964;N:143263	51				128538964	164901356	144434115	190840964	143263	SRX28685521	SRS24952425	SRA2124446	SARL, Environmental and Molecular Toxicology, Oregon State University	SARL, Environmental and Molecular Toxicology, Oregon State University												B		usable mapping rate	illumina	novaseq_era	unknown	other	unknown	bulk	unknown	unknown	United States	2025-05-06	Multi-stage	Embryo	Whole Organism	All anatomical structures
36062	36062	SRR33444774	SRX28685520	SRS24952424	SRP583274	PRJNA1259243	Structure Dependent Developmental Toxicity of Alkyl Substituted Naphthalenes	GSE296410	Transcriptome Analysis	Naphthalene and its alkyl substituted derivatives are among the most abundant polycyclic aromatic hydrocarbons PAHs in environmental and human exposure studies yet their developmental toxicity and mode of action remain poorly understood due to challenges in testing of semi volatile compounds. This study developed a vial based high through put method to effectively assess the activity of naphthalenes and a set of 24 alkyl substituted naphthalenes. Early life stage zebrafish were exposed to a concentration series of each chemical 0 50 µM in rotating sealed glass vials to minimize volatilization. Benchmark concentration BMC50 values were calculated for morphological endpoints and lowest effect levels were determined for behavioral effects. The data were assessed for evidence of a narcotic mode of action using body burden measurements for select chemicals and logKow modeling. Targeted transcriptomics at a single concentration and timepoint as well as in silico molecular docking were conducted to generate mode of action hypothesis. The vial method enabled detection of highly variable developmental toxicity not previously observed using standard 96 well plate exposures. LogKow and body burden were poor predictors of toxicity suggesting a non narcotic mode of action. Transcriptomic analysis revealed a limited but notable set of differentially expressed genes with evidence for the disruption of glucocorticoid signaling pathways. Molecular docking identified potential protein targets e.g. CYP1A2 NT5E FOLR1 that may mediate observed effects. This study demonstrates the importance of appropriate exposure methods for semi volatile compounds reveals structure dependent toxicity among alkyl substituted naphthalenes and provides a foundation for further mechanistic studies and improved risk assessment of alkyl substituted PAHs. Overall design: Zebrafish were exposed to 25 chemicals naphthalene and 24 alkyl sustituted naphthalenes using a vial exposure method. Embryos were exposed to 20 uM of each chemical in groups of…				1 6 7 trimethylnaphthalene B	GSM8969927		source name:whole animal\|tissue:whole animal\|genotype:5D WT\|treatment:1 6 7 trimethylnaphthalene\|batch:1\|geo loc name:missing\|collection date:missing	1 6 7 trimethylnaphthalene B	Demultiplexing and alignment was performed using the manufacture provided processing platform TempO SeqR BioSpyder Technologies Inc. Carlsbad CA United States. Counts of genes with multiple probes were summed as instructed by the manufacturer resulting in 3112 unique genes Read counts were normalized by upper quartile normalization using the calcNormFactors function from the Bioconductor package in EdgeR using the 75th percentile of reads per samples as the scaling factor. Principal component analysis PCA of normalized read counts was used to determine that a batch effect was present based on exposure and RNA extraction day nested by experimental design. At this time one control sample control day1 B was identified as an outlier and removed from all future analysis. The batch effect was removed from unnormalized data using the ComBat seq function from the SVA R package Normalzied read counts were normalized by upper quartile normalization using the calcNormFactors function from the Bioconductor package in EdgeR using the 75th percentile of reads per samples as the scaling factor. Assembly: GRCz11 Supplementary files format and content: .csv file containing raw count data Supplementary files format and content: .csv file containing batch corrected and normalized data	whole animal	When the embryos were 6hpf they were bleached in groups of 1000 or less. Embryos were placed in a strainer for transfer between solutions in 500 mL containers. First embryos were placed in system fish water for 1 minute then transferred to a 0.5% sodium chloride solution for 5 minutes then transferred to a % sodium hypochlorite solution for 1 minute and rinsed in EM for 5 minutes and transferred to a clean glass petri dish. Embryos were then loaded into 2mL glass exposure vials in groups of 8 using a glass pasture pipette. Excess EM was removed and 2mL of fresh EM was immediately added. Chemical stocks were diluted to 200 times the desired exposure concentration of 20 µM using DMSO. 10 µL of diluted chemical stock was added to each vial vials were immediately capped and inverted 3 times to ensure mixing no precipitate was observed for any chemicals at the concentrations tested. Vials were loaded into a custom 3D printed vial holder for slow end over end rotation in a dark 28°C incubator overnight. At 24 hours embryos were removed from exposure vials and plated into 96 well plates loaded with 100 µL clean EM. At 48 hpf embryos were collected for RNA extraction embryos from two exposure vials were kept as holdback for morphological assessment. From the remaining wells 6 phenotypically normal embryos were collected and pooled in their vial groups into 1.5 mL Eppendorf tubes placed on ice 30 sec excess media removed placed back on ice 30 sec and 200 µL RNA shield added. Five pooled samples were collected from each treatment.	1mL of 1µm beads and 500µL of lysis buffer were loaded into a round bottom 2mL 96 well plate. 100µL of lysis buffer was added to tube containing pooled embryo samples and 200µL of RNA shield the full contents of the tube were then transferred to the homogenization plate. The plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 275 µL of the supernatant was then transferred to two plates containing 275µL of ethanol. RNA extraction was performed using the KingFisher Apex system. Briefly this process uses Mag beads to collect RNA from the sample post series of washes the RNA is eluted in 50µLof ultra pure water. RNA quality was assessed using the Agilent 4150 TapeStation System. All samples used had RIN scores >9.8. RNA concentration was determined using ThermoFisher Quant iT RNA Reagent and assay kit and ThermoFisher Varioskan ALF multimode plate reader in triplicate samples with high variability between reads were repeated in triplicate. The average of the three quantification reads was used for RNA concentration normalization. Each treatment had 4 5 viable RNA extraction replicates for treatments with 5 viable samples the 4 with the highest concentration and RIN score were used 3 control samples were used from each day group. All 94 samples were normalized to the lowest concentration sample 14.9ng/µL. 1 µL per sample of normalized RNA was used as input for library preparation following manufactures protocol for the 96 sample TempO Seq S1500 + Zebrafish Surrogate Assay panel BioSpyder Technologies Inc. Carlsbad CA United States. Briefly samples were added to the assay plate with an equal volume of 2x enhanced lysis buffer and processed through annealing with detector oligos digestion and ligation. Proper amplification was verified using real time PCR green fluorescence. Library purification was performed using the Macherey Nagel NucleoSpin Gel and PCR Cleanup Kit following adjustments detailed in the manufacturer’s instructions. PCR amplification products were pooled into a s…		tissue:whole animal\|genotype:5D WT\|treatment:1 6 7 trimethylnaphthalene\|batch:1	GSM8969927	GSM8969927: 1 6 7 trimethylnaphthalene B; Danio rerio; OTHER	GSM8969927 r1	GSM8969927	1	1mL of 1µm beads and 500µL of lysis buffer were loaded into a round bottom 2mL 96 well plate. 100µL of lysis buffer was added to tube containing pooled embryo samples and 200µL of RNA shield the full contents of the tube were then transferred to the homogenization plate. The plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 275 µL of the supernatant was then transferred to two plates containing 275µL of ethanol. RNA extraction was performed using the KingFisher Apex system. Briefly this process uses Mag beads to collect RNA from the sample post series of washes the RNA is eluted in 50µLof ultra pure water. RNA quality was assessed using the Agilent 4150 TapeStation System. All samples used had RIN scores >9.8. RNA concentration was determined using ThermoFisher Quant iT RNA Reagent and assay kit and ThermoFisher Varioskan ALF multimode plate reader in triplicate samples with high variability between reads were repeated in triplicate. The average of the three quantification reads was used for RNA concentration normalization. Each treatment had 4 5 viable RNA extraction replicates for treatments with 5 viable samples the 4 with the highest concentration and RIN score were used 3 control samples were used from each day group. All 94 samples were normalized to the lowest concentration sample 14.9ng/µL. 1 µL per sample of normalized RNA was used as input for library preparation following manufactures protocol for the 96 sample TempO Seq S1500 + Zebrafish Surrogate Assay panel BioSpyder Technologies Inc. Carlsbad CA United States. Briefly samples were added to the assay plate with an equal volume of 2x enhanced lysis buffer and processed through annealing with detector oligos digestion and ligation. Proper amplification was verified using real time PCR green fluorescence. Library purification was performed using the Macherey Nagel NucleoSpin Gel and PCR Cleanup Kit following adjustments detailed in the manufacturer's instructions. PCR amplification products were pooled into a s…	OTHER	TRANSCRIPTOMIC	other	SINGLE	ILLUMINA	Illumina NovaSeq X	SRP583274			9_B.fastq.gz	fastq	550098036.0	10786236.0	GSM8969927 r1	0:51	A:112188300;C:144676062;G:126419391;T:166689819;N:124464	51				112188300	144676062	126419391	166689819	124464	SRX28685520	SRS24952424	SRA2124446	SARL, Environmental and Molecular Toxicology, Oregon State University	SARL, Environmental and Molecular Toxicology, Oregon State University												B		usable mapping rate	illumina	novaseq_era	unknown	other	unknown	bulk	unknown	unknown	United States	2025-05-06	Multi-stage	Embryo	Whole Organism	All anatomical structures
36063	36063	SRR33444775	SRX28685519	SRS24952422	SRP583274	PRJNA1259243	Structure Dependent Developmental Toxicity of Alkyl Substituted Naphthalenes	GSE296410	Transcriptome Analysis	Naphthalene and its alkyl substituted derivatives are among the most abundant polycyclic aromatic hydrocarbons PAHs in environmental and human exposure studies yet their developmental toxicity and mode of action remain poorly understood due to challenges in testing of semi volatile compounds. This study developed a vial based high through put method to effectively assess the activity of naphthalenes and a set of 24 alkyl substituted naphthalenes. Early life stage zebrafish were exposed to a concentration series of each chemical 0 50 µM in rotating sealed glass vials to minimize volatilization. Benchmark concentration BMC50 values were calculated for morphological endpoints and lowest effect levels were determined for behavioral effects. The data were assessed for evidence of a narcotic mode of action using body burden measurements for select chemicals and logKow modeling. Targeted transcriptomics at a single concentration and timepoint as well as in silico molecular docking were conducted to generate mode of action hypothesis. The vial method enabled detection of highly variable developmental toxicity not previously observed using standard 96 well plate exposures. LogKow and body burden were poor predictors of toxicity suggesting a non narcotic mode of action. Transcriptomic analysis revealed a limited but notable set of differentially expressed genes with evidence for the disruption of glucocorticoid signaling pathways. Molecular docking identified potential protein targets e.g. CYP1A2 NT5E FOLR1 that may mediate observed effects. This study demonstrates the importance of appropriate exposure methods for semi volatile compounds reveals structure dependent toxicity among alkyl substituted naphthalenes and provides a foundation for further mechanistic studies and improved risk assessment of alkyl substituted PAHs. Overall design: Zebrafish were exposed to 25 chemicals naphthalene and 24 alkyl sustituted naphthalenes using a vial exposure method. Embryos were exposed to 20 uM of each chemical in groups of…				1 6 7 trimethylnaphthalene A	GSM8969926		source name:whole animal\|tissue:whole animal\|genotype:5D WT\|treatment:1 6 7 trimethylnaphthalene\|batch:1\|geo loc name:missing\|collection date:missing	1 6 7 trimethylnaphthalene A	Demultiplexing and alignment was performed using the manufacture provided processing platform TempO SeqR BioSpyder Technologies Inc. Carlsbad CA United States. Counts of genes with multiple probes were summed as instructed by the manufacturer resulting in 3112 unique genes Read counts were normalized by upper quartile normalization using the calcNormFactors function from the Bioconductor package in EdgeR using the 75th percentile of reads per samples as the scaling factor. Principal component analysis PCA of normalized read counts was used to determine that a batch effect was present based on exposure and RNA extraction day nested by experimental design. At this time one control sample control day1 B was identified as an outlier and removed from all future analysis. The batch effect was removed from unnormalized data using the ComBat seq function from the SVA R package Normalzied read counts were normalized by upper quartile normalization using the calcNormFactors function from the Bioconductor package in EdgeR using the 75th percentile of reads per samples as the scaling factor. Assembly: GRCz11 Supplementary files format and content: .csv file containing raw count data Supplementary files format and content: .csv file containing batch corrected and normalized data	whole animal	When the embryos were 6hpf they were bleached in groups of 1000 or less. Embryos were placed in a strainer for transfer between solutions in 500 mL containers. First embryos were placed in system fish water for 1 minute then transferred to a 0.5% sodium chloride solution for 5 minutes then transferred to a % sodium hypochlorite solution for 1 minute and rinsed in EM for 5 minutes and transferred to a clean glass petri dish. Embryos were then loaded into 2mL glass exposure vials in groups of 8 using a glass pasture pipette. Excess EM was removed and 2mL of fresh EM was immediately added. Chemical stocks were diluted to 200 times the desired exposure concentration of 20 µM using DMSO. 10 µL of diluted chemical stock was added to each vial vials were immediately capped and inverted 3 times to ensure mixing no precipitate was observed for any chemicals at the concentrations tested. Vials were loaded into a custom 3D printed vial holder for slow end over end rotation in a dark 28°C incubator overnight. At 24 hours embryos were removed from exposure vials and plated into 96 well plates loaded with 100 µL clean EM. At 48 hpf embryos were collected for RNA extraction embryos from two exposure vials were kept as holdback for morphological assessment. From the remaining wells 6 phenotypically normal embryos were collected and pooled in their vial groups into 1.5 mL Eppendorf tubes placed on ice 30 sec excess media removed placed back on ice 30 sec and 200 µL RNA shield added. Five pooled samples were collected from each treatment.	1mL of 1µm beads and 500µL of lysis buffer were loaded into a round bottom 2mL 96 well plate. 100µL of lysis buffer was added to tube containing pooled embryo samples and 200µL of RNA shield the full contents of the tube were then transferred to the homogenization plate. The plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 275 µL of the supernatant was then transferred to two plates containing 275µL of ethanol. RNA extraction was performed using the KingFisher Apex system. Briefly this process uses Mag beads to collect RNA from the sample post series of washes the RNA is eluted in 50µLof ultra pure water. RNA quality was assessed using the Agilent 4150 TapeStation System. All samples used had RIN scores >9.8. RNA concentration was determined using ThermoFisher Quant iT RNA Reagent and assay kit and ThermoFisher Varioskan ALF multimode plate reader in triplicate samples with high variability between reads were repeated in triplicate. The average of the three quantification reads was used for RNA concentration normalization. Each treatment had 4 5 viable RNA extraction replicates for treatments with 5 viable samples the 4 with the highest concentration and RIN score were used 3 control samples were used from each day group. All 94 samples were normalized to the lowest concentration sample 14.9ng/µL. 1 µL per sample of normalized RNA was used as input for library preparation following manufactures protocol for the 96 sample TempO Seq S1500 + Zebrafish Surrogate Assay panel BioSpyder Technologies Inc. Carlsbad CA United States. Briefly samples were added to the assay plate with an equal volume of 2x enhanced lysis buffer and processed through annealing with detector oligos digestion and ligation. Proper amplification was verified using real time PCR green fluorescence. Library purification was performed using the Macherey Nagel NucleoSpin Gel and PCR Cleanup Kit following adjustments detailed in the manufacturer’s instructions. PCR amplification products were pooled into a s…		tissue:whole animal\|genotype:5D WT\|treatment:1 6 7 trimethylnaphthalene\|batch:1	GSM8969926	GSM8969926: 1 6 7 trimethylnaphthalene A; Danio rerio; OTHER	GSM8969926 r1	GSM8969926	1	1mL of 1µm beads and 500µL of lysis buffer were loaded into a round bottom 2mL 96 well plate. 100µL of lysis buffer was added to tube containing pooled embryo samples and 200µL of RNA shield the full contents of the tube were then transferred to the homogenization plate. The plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 275 µL of the supernatant was then transferred to two plates containing 275µL of ethanol. RNA extraction was performed using the KingFisher Apex system. Briefly this process uses Mag beads to collect RNA from the sample post series of washes the RNA is eluted in 50µLof ultra pure water. RNA quality was assessed using the Agilent 4150 TapeStation System. All samples used had RIN scores >9.8. RNA concentration was determined using ThermoFisher Quant iT RNA Reagent and assay kit and ThermoFisher Varioskan ALF multimode plate reader in triplicate samples with high variability between reads were repeated in triplicate. The average of the three quantification reads was used for RNA concentration normalization. Each treatment had 4 5 viable RNA extraction replicates for treatments with 5 viable samples the 4 with the highest concentration and RIN score were used 3 control samples were used from each day group. All 94 samples were normalized to the lowest concentration sample 14.9ng/µL. 1 µL per sample of normalized RNA was used as input for library preparation following manufactures protocol for the 96 sample TempO Seq S1500 + Zebrafish Surrogate Assay panel BioSpyder Technologies Inc. Carlsbad CA United States. Briefly samples were added to the assay plate with an equal volume of 2x enhanced lysis buffer and processed through annealing with detector oligos digestion and ligation. Proper amplification was verified using real time PCR green fluorescence. Library purification was performed using the Macherey Nagel NucleoSpin Gel and PCR Cleanup Kit following adjustments detailed in the manufacturer's instructions. PCR amplification products were pooled into a s…	OTHER	TRANSCRIPTOMIC	other	SINGLE	ILLUMINA	Illumina NovaSeq X	SRP583274			9_A.fastq.gz	fastq	449907771.0	8821721.0	GSM8969926 r1	0:51	A:91463949;C:117779693;G:103518447;T:137043427;N:102255	51				91463949	117779693	103518447	137043427	102255	SRX28685519	SRS24952422	SRA2124446	SARL, Environmental and Molecular Toxicology, Oregon State University	SARL, Environmental and Molecular Toxicology, Oregon State University												B		usable mapping rate	illumina	novaseq_era	unknown	other	unknown	bulk	unknown	unknown	United States	2025-05-06	Multi-stage	Embryo	Whole Organism	All anatomical structures
36064	36064	SRR33444776	SRX28685518	SRS24952421	SRP583274	PRJNA1259243	Structure Dependent Developmental Toxicity of Alkyl Substituted Naphthalenes	GSE296410	Transcriptome Analysis	Naphthalene and its alkyl substituted derivatives are among the most abundant polycyclic aromatic hydrocarbons PAHs in environmental and human exposure studies yet their developmental toxicity and mode of action remain poorly understood due to challenges in testing of semi volatile compounds. This study developed a vial based high through put method to effectively assess the activity of naphthalenes and a set of 24 alkyl substituted naphthalenes. Early life stage zebrafish were exposed to a concentration series of each chemical 0 50 µM in rotating sealed glass vials to minimize volatilization. Benchmark concentration BMC50 values were calculated for morphological endpoints and lowest effect levels were determined for behavioral effects. The data were assessed for evidence of a narcotic mode of action using body burden measurements for select chemicals and logKow modeling. Targeted transcriptomics at a single concentration and timepoint as well as in silico molecular docking were conducted to generate mode of action hypothesis. The vial method enabled detection of highly variable developmental toxicity not previously observed using standard 96 well plate exposures. LogKow and body burden were poor predictors of toxicity suggesting a non narcotic mode of action. Transcriptomic analysis revealed a limited but notable set of differentially expressed genes with evidence for the disruption of glucocorticoid signaling pathways. Molecular docking identified potential protein targets e.g. CYP1A2 NT5E FOLR1 that may mediate observed effects. This study demonstrates the importance of appropriate exposure methods for semi volatile compounds reveals structure dependent toxicity among alkyl substituted naphthalenes and provides a foundation for further mechanistic studies and improved risk assessment of alkyl substituted PAHs. Overall design: Zebrafish were exposed to 25 chemicals naphthalene and 24 alkyl sustituted naphthalenes using a vial exposure method. Embryos were exposed to 20 uM of each chemical in groups of…				1 ethylnaphthalene E	GSM8969925		source name:whole animal\|tissue:whole animal\|genotype:5D WT\|treatment:1 ethylnaphthalene\|batch:1\|geo loc name:missing\|collection date:missing	1 ethylnaphthalene E	Demultiplexing and alignment was performed using the manufacture provided processing platform TempO SeqR BioSpyder Technologies Inc. Carlsbad CA United States. Counts of genes with multiple probes were summed as instructed by the manufacturer resulting in 3112 unique genes Read counts were normalized by upper quartile normalization using the calcNormFactors function from the Bioconductor package in EdgeR using the 75th percentile of reads per samples as the scaling factor. Principal component analysis PCA of normalized read counts was used to determine that a batch effect was present based on exposure and RNA extraction day nested by experimental design. At this time one control sample control day1 B was identified as an outlier and removed from all future analysis. The batch effect was removed from unnormalized data using the ComBat seq function from the SVA R package Normalzied read counts were normalized by upper quartile normalization using the calcNormFactors function from the Bioconductor package in EdgeR using the 75th percentile of reads per samples as the scaling factor. Assembly: GRCz11 Supplementary files format and content: .csv file containing raw count data Supplementary files format and content: .csv file containing batch corrected and normalized data	whole animal	When the embryos were 6hpf they were bleached in groups of 1000 or less. Embryos were placed in a strainer for transfer between solutions in 500 mL containers. First embryos were placed in system fish water for 1 minute then transferred to a 0.5% sodium chloride solution for 5 minutes then transferred to a % sodium hypochlorite solution for 1 minute and rinsed in EM for 5 minutes and transferred to a clean glass petri dish. Embryos were then loaded into 2mL glass exposure vials in groups of 8 using a glass pasture pipette. Excess EM was removed and 2mL of fresh EM was immediately added. Chemical stocks were diluted to 200 times the desired exposure concentration of 20 µM using DMSO. 10 µL of diluted chemical stock was added to each vial vials were immediately capped and inverted 3 times to ensure mixing no precipitate was observed for any chemicals at the concentrations tested. Vials were loaded into a custom 3D printed vial holder for slow end over end rotation in a dark 28°C incubator overnight. At 24 hours embryos were removed from exposure vials and plated into 96 well plates loaded with 100 µL clean EM. At 48 hpf embryos were collected for RNA extraction embryos from two exposure vials were kept as holdback for morphological assessment. From the remaining wells 6 phenotypically normal embryos were collected and pooled in their vial groups into 1.5 mL Eppendorf tubes placed on ice 30 sec excess media removed placed back on ice 30 sec and 200 µL RNA shield added. Five pooled samples were collected from each treatment.	1mL of 1µm beads and 500µL of lysis buffer were loaded into a round bottom 2mL 96 well plate. 100µL of lysis buffer was added to tube containing pooled embryo samples and 200µL of RNA shield the full contents of the tube were then transferred to the homogenization plate. The plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 275 µL of the supernatant was then transferred to two plates containing 275µL of ethanol. RNA extraction was performed using the KingFisher Apex system. Briefly this process uses Mag beads to collect RNA from the sample post series of washes the RNA is eluted in 50µLof ultra pure water. RNA quality was assessed using the Agilent 4150 TapeStation System. All samples used had RIN scores >9.8. RNA concentration was determined using ThermoFisher Quant iT RNA Reagent and assay kit and ThermoFisher Varioskan ALF multimode plate reader in triplicate samples with high variability between reads were repeated in triplicate. The average of the three quantification reads was used for RNA concentration normalization. Each treatment had 4 5 viable RNA extraction replicates for treatments with 5 viable samples the 4 with the highest concentration and RIN score were used 3 control samples were used from each day group. All 94 samples were normalized to the lowest concentration sample 14.9ng/µL. 1 µL per sample of normalized RNA was used as input for library preparation following manufactures protocol for the 96 sample TempO Seq S1500 + Zebrafish Surrogate Assay panel BioSpyder Technologies Inc. Carlsbad CA United States. Briefly samples were added to the assay plate with an equal volume of 2x enhanced lysis buffer and processed through annealing with detector oligos digestion and ligation. Proper amplification was verified using real time PCR green fluorescence. Library purification was performed using the Macherey Nagel NucleoSpin Gel and PCR Cleanup Kit following adjustments detailed in the manufacturer’s instructions. PCR amplification products were pooled into a s…		tissue:whole animal\|genotype:5D WT\|treatment:1 ethylnaphthalene\|batch:1	GSM8969925	GSM8969925: 1 ethylnaphthalene E; Danio rerio; OTHER	GSM8969925 r1	GSM8969925	1	1mL of 1µm beads and 500µL of lysis buffer were loaded into a round bottom 2mL 96 well plate. 100µL of lysis buffer was added to tube containing pooled embryo samples and 200µL of RNA shield the full contents of the tube were then transferred to the homogenization plate. The plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 275 µL of the supernatant was then transferred to two plates containing 275µL of ethanol. RNA extraction was performed using the KingFisher Apex system. Briefly this process uses Mag beads to collect RNA from the sample post series of washes the RNA is eluted in 50µLof ultra pure water. RNA quality was assessed using the Agilent 4150 TapeStation System. All samples used had RIN scores >9.8. RNA concentration was determined using ThermoFisher Quant iT RNA Reagent and assay kit and ThermoFisher Varioskan ALF multimode plate reader in triplicate samples with high variability between reads were repeated in triplicate. The average of the three quantification reads was used for RNA concentration normalization. Each treatment had 4 5 viable RNA extraction replicates for treatments with 5 viable samples the 4 with the highest concentration and RIN score were used 3 control samples were used from each day group. All 94 samples were normalized to the lowest concentration sample 14.9ng/µL. 1 µL per sample of normalized RNA was used as input for library preparation following manufactures protocol for the 96 sample TempO Seq S1500 + Zebrafish Surrogate Assay panel BioSpyder Technologies Inc. Carlsbad CA United States. Briefly samples were added to the assay plate with an equal volume of 2x enhanced lysis buffer and processed through annealing with detector oligos digestion and ligation. Proper amplification was verified using real time PCR green fluorescence. Library purification was performed using the Macherey Nagel NucleoSpin Gel and PCR Cleanup Kit following adjustments detailed in the manufacturer's instructions. PCR amplification products were pooled into a s…	OTHER	TRANSCRIPTOMIC	other	SINGLE	ILLUMINA	Illumina NovaSeq X	SRP583274			8_E.fastq.gz	fastq	596647062.0	11698962.0	GSM8969925 r1	0:51	A:121970662;C:156141284;G:138301387;T:180098649;N:135080	51				121970662	156141284	138301387	180098649	135080	SRX28685518	SRS24952421	SRA2124446	SARL, Environmental and Molecular Toxicology, Oregon State University	SARL, Environmental and Molecular Toxicology, Oregon State University												B		usable mapping rate	illumina	novaseq_era	unknown	other	unknown	bulk	unknown	unknown	United States	2025-05-06	Multi-stage	Embryo	Whole Organism	All anatomical structures
36065	36065	SRR33444777	SRX28685517	SRS24952420	SRP583274	PRJNA1259243	Structure Dependent Developmental Toxicity of Alkyl Substituted Naphthalenes	GSE296410	Transcriptome Analysis	Naphthalene and its alkyl substituted derivatives are among the most abundant polycyclic aromatic hydrocarbons PAHs in environmental and human exposure studies yet their developmental toxicity and mode of action remain poorly understood due to challenges in testing of semi volatile compounds. This study developed a vial based high through put method to effectively assess the activity of naphthalenes and a set of 24 alkyl substituted naphthalenes. Early life stage zebrafish were exposed to a concentration series of each chemical 0 50 µM in rotating sealed glass vials to minimize volatilization. Benchmark concentration BMC50 values were calculated for morphological endpoints and lowest effect levels were determined for behavioral effects. The data were assessed for evidence of a narcotic mode of action using body burden measurements for select chemicals and logKow modeling. Targeted transcriptomics at a single concentration and timepoint as well as in silico molecular docking were conducted to generate mode of action hypothesis. The vial method enabled detection of highly variable developmental toxicity not previously observed using standard 96 well plate exposures. LogKow and body burden were poor predictors of toxicity suggesting a non narcotic mode of action. Transcriptomic analysis revealed a limited but notable set of differentially expressed genes with evidence for the disruption of glucocorticoid signaling pathways. Molecular docking identified potential protein targets e.g. CYP1A2 NT5E FOLR1 that may mediate observed effects. This study demonstrates the importance of appropriate exposure methods for semi volatile compounds reveals structure dependent toxicity among alkyl substituted naphthalenes and provides a foundation for further mechanistic studies and improved risk assessment of alkyl substituted PAHs. Overall design: Zebrafish were exposed to 25 chemicals naphthalene and 24 alkyl sustituted naphthalenes using a vial exposure method. Embryos were exposed to 20 uM of each chemical in groups of…				1 ethylnaphthalene C	GSM8969924		source name:whole animal\|tissue:whole animal\|genotype:5D WT\|treatment:1 ethylnaphthalene\|batch:1\|geo loc name:missing\|collection date:missing	1 ethylnaphthalene C	Demultiplexing and alignment was performed using the manufacture provided processing platform TempO SeqR BioSpyder Technologies Inc. Carlsbad CA United States. Counts of genes with multiple probes were summed as instructed by the manufacturer resulting in 3112 unique genes Read counts were normalized by upper quartile normalization using the calcNormFactors function from the Bioconductor package in EdgeR using the 75th percentile of reads per samples as the scaling factor. Principal component analysis PCA of normalized read counts was used to determine that a batch effect was present based on exposure and RNA extraction day nested by experimental design. At this time one control sample control day1 B was identified as an outlier and removed from all future analysis. The batch effect was removed from unnormalized data using the ComBat seq function from the SVA R package Normalzied read counts were normalized by upper quartile normalization using the calcNormFactors function from the Bioconductor package in EdgeR using the 75th percentile of reads per samples as the scaling factor. Assembly: GRCz11 Supplementary files format and content: .csv file containing raw count data Supplementary files format and content: .csv file containing batch corrected and normalized data	whole animal	When the embryos were 6hpf they were bleached in groups of 1000 or less. Embryos were placed in a strainer for transfer between solutions in 500 mL containers. First embryos were placed in system fish water for 1 minute then transferred to a 0.5% sodium chloride solution for 5 minutes then transferred to a % sodium hypochlorite solution for 1 minute and rinsed in EM for 5 minutes and transferred to a clean glass petri dish. Embryos were then loaded into 2mL glass exposure vials in groups of 8 using a glass pasture pipette. Excess EM was removed and 2mL of fresh EM was immediately added. Chemical stocks were diluted to 200 times the desired exposure concentration of 20 µM using DMSO. 10 µL of diluted chemical stock was added to each vial vials were immediately capped and inverted 3 times to ensure mixing no precipitate was observed for any chemicals at the concentrations tested. Vials were loaded into a custom 3D printed vial holder for slow end over end rotation in a dark 28°C incubator overnight. At 24 hours embryos were removed from exposure vials and plated into 96 well plates loaded with 100 µL clean EM. At 48 hpf embryos were collected for RNA extraction embryos from two exposure vials were kept as holdback for morphological assessment. From the remaining wells 6 phenotypically normal embryos were collected and pooled in their vial groups into 1.5 mL Eppendorf tubes placed on ice 30 sec excess media removed placed back on ice 30 sec and 200 µL RNA shield added. Five pooled samples were collected from each treatment.	1mL of 1µm beads and 500µL of lysis buffer were loaded into a round bottom 2mL 96 well plate. 100µL of lysis buffer was added to tube containing pooled embryo samples and 200µL of RNA shield the full contents of the tube were then transferred to the homogenization plate. The plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 275 µL of the supernatant was then transferred to two plates containing 275µL of ethanol. RNA extraction was performed using the KingFisher Apex system. Briefly this process uses Mag beads to collect RNA from the sample post series of washes the RNA is eluted in 50µLof ultra pure water. RNA quality was assessed using the Agilent 4150 TapeStation System. All samples used had RIN scores >9.8. RNA concentration was determined using ThermoFisher Quant iT RNA Reagent and assay kit and ThermoFisher Varioskan ALF multimode plate reader in triplicate samples with high variability between reads were repeated in triplicate. The average of the three quantification reads was used for RNA concentration normalization. Each treatment had 4 5 viable RNA extraction replicates for treatments with 5 viable samples the 4 with the highest concentration and RIN score were used 3 control samples were used from each day group. All 94 samples were normalized to the lowest concentration sample 14.9ng/µL. 1 µL per sample of normalized RNA was used as input for library preparation following manufactures protocol for the 96 sample TempO Seq S1500 + Zebrafish Surrogate Assay panel BioSpyder Technologies Inc. Carlsbad CA United States. Briefly samples were added to the assay plate with an equal volume of 2x enhanced lysis buffer and processed through annealing with detector oligos digestion and ligation. Proper amplification was verified using real time PCR green fluorescence. Library purification was performed using the Macherey Nagel NucleoSpin Gel and PCR Cleanup Kit following adjustments detailed in the manufacturer’s instructions. PCR amplification products were pooled into a s…		tissue:whole animal\|genotype:5D WT\|treatment:1 ethylnaphthalene\|batch:1	GSM8969924	GSM8969924: 1 ethylnaphthalene C; Danio rerio; OTHER	GSM8969924 r1	GSM8969924	1	1mL of 1µm beads and 500µL of lysis buffer were loaded into a round bottom 2mL 96 well plate. 100µL of lysis buffer was added to tube containing pooled embryo samples and 200µL of RNA shield the full contents of the tube were then transferred to the homogenization plate. The plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 275 µL of the supernatant was then transferred to two plates containing 275µL of ethanol. RNA extraction was performed using the KingFisher Apex system. Briefly this process uses Mag beads to collect RNA from the sample post series of washes the RNA is eluted in 50µLof ultra pure water. RNA quality was assessed using the Agilent 4150 TapeStation System. All samples used had RIN scores >9.8. RNA concentration was determined using ThermoFisher Quant iT RNA Reagent and assay kit and ThermoFisher Varioskan ALF multimode plate reader in triplicate samples with high variability between reads were repeated in triplicate. The average of the three quantification reads was used for RNA concentration normalization. Each treatment had 4 5 viable RNA extraction replicates for treatments with 5 viable samples the 4 with the highest concentration and RIN score were used 3 control samples were used from each day group. All 94 samples were normalized to the lowest concentration sample 14.9ng/µL. 1 µL per sample of normalized RNA was used as input for library preparation following manufactures protocol for the 96 sample TempO Seq S1500 + Zebrafish Surrogate Assay panel BioSpyder Technologies Inc. Carlsbad CA United States. Briefly samples were added to the assay plate with an equal volume of 2x enhanced lysis buffer and processed through annealing with detector oligos digestion and ligation. Proper amplification was verified using real time PCR green fluorescence. Library purification was performed using the Macherey Nagel NucleoSpin Gel and PCR Cleanup Kit following adjustments detailed in the manufacturer's instructions. PCR amplification products were pooled into a s…	OTHER	TRANSCRIPTOMIC	other	SINGLE	ILLUMINA	Illumina NovaSeq X	SRP583274			8_C.fastq.gz	fastq	659636703.0	12934053.0	GSM8969924 r1	0:51	A:134295377;C:173327337;G:153006654;T:198857895;N:149440	51				134295377	173327337	153006654	198857895	149440	SRX28685517	SRS24952420	SRA2124446	SARL, Environmental and Molecular Toxicology, Oregon State University	SARL, Environmental and Molecular Toxicology, Oregon State University												B		usable mapping rate	illumina	novaseq_era	unknown	other	unknown	bulk	unknown	unknown	United States	2025-05-06	Multi-stage	Embryo	Whole Organism	All anatomical structures
36066	36066	SRR33444778	SRX28685516	SRS24952418	SRP583274	PRJNA1259243	Structure Dependent Developmental Toxicity of Alkyl Substituted Naphthalenes	GSE296410	Transcriptome Analysis	Naphthalene and its alkyl substituted derivatives are among the most abundant polycyclic aromatic hydrocarbons PAHs in environmental and human exposure studies yet their developmental toxicity and mode of action remain poorly understood due to challenges in testing of semi volatile compounds. This study developed a vial based high through put method to effectively assess the activity of naphthalenes and a set of 24 alkyl substituted naphthalenes. Early life stage zebrafish were exposed to a concentration series of each chemical 0 50 µM in rotating sealed glass vials to minimize volatilization. Benchmark concentration BMC50 values were calculated for morphological endpoints and lowest effect levels were determined for behavioral effects. The data were assessed for evidence of a narcotic mode of action using body burden measurements for select chemicals and logKow modeling. Targeted transcriptomics at a single concentration and timepoint as well as in silico molecular docking were conducted to generate mode of action hypothesis. The vial method enabled detection of highly variable developmental toxicity not previously observed using standard 96 well plate exposures. LogKow and body burden were poor predictors of toxicity suggesting a non narcotic mode of action. Transcriptomic analysis revealed a limited but notable set of differentially expressed genes with evidence for the disruption of glucocorticoid signaling pathways. Molecular docking identified potential protein targets e.g. CYP1A2 NT5E FOLR1 that may mediate observed effects. This study demonstrates the importance of appropriate exposure methods for semi volatile compounds reveals structure dependent toxicity among alkyl substituted naphthalenes and provides a foundation for further mechanistic studies and improved risk assessment of alkyl substituted PAHs. Overall design: Zebrafish were exposed to 25 chemicals naphthalene and 24 alkyl sustituted naphthalenes using a vial exposure method. Embryos were exposed to 20 uM of each chemical in groups of…				1 ethylnaphthalene B	GSM8969923		source name:whole animal\|tissue:whole animal\|genotype:5D WT\|treatment:1 ethylnaphthalene\|batch:1\|geo loc name:missing\|collection date:missing	1 ethylnaphthalene B	Demultiplexing and alignment was performed using the manufacture provided processing platform TempO SeqR BioSpyder Technologies Inc. Carlsbad CA United States. Counts of genes with multiple probes were summed as instructed by the manufacturer resulting in 3112 unique genes Read counts were normalized by upper quartile normalization using the calcNormFactors function from the Bioconductor package in EdgeR using the 75th percentile of reads per samples as the scaling factor. Principal component analysis PCA of normalized read counts was used to determine that a batch effect was present based on exposure and RNA extraction day nested by experimental design. At this time one control sample control day1 B was identified as an outlier and removed from all future analysis. The batch effect was removed from unnormalized data using the ComBat seq function from the SVA R package Normalzied read counts were normalized by upper quartile normalization using the calcNormFactors function from the Bioconductor package in EdgeR using the 75th percentile of reads per samples as the scaling factor. Assembly: GRCz11 Supplementary files format and content: .csv file containing raw count data Supplementary files format and content: .csv file containing batch corrected and normalized data	whole animal	When the embryos were 6hpf they were bleached in groups of 1000 or less. Embryos were placed in a strainer for transfer between solutions in 500 mL containers. First embryos were placed in system fish water for 1 minute then transferred to a 0.5% sodium chloride solution for 5 minutes then transferred to a % sodium hypochlorite solution for 1 minute and rinsed in EM for 5 minutes and transferred to a clean glass petri dish. Embryos were then loaded into 2mL glass exposure vials in groups of 8 using a glass pasture pipette. Excess EM was removed and 2mL of fresh EM was immediately added. Chemical stocks were diluted to 200 times the desired exposure concentration of 20 µM using DMSO. 10 µL of diluted chemical stock was added to each vial vials were immediately capped and inverted 3 times to ensure mixing no precipitate was observed for any chemicals at the concentrations tested. Vials were loaded into a custom 3D printed vial holder for slow end over end rotation in a dark 28°C incubator overnight. At 24 hours embryos were removed from exposure vials and plated into 96 well plates loaded with 100 µL clean EM. At 48 hpf embryos were collected for RNA extraction embryos from two exposure vials were kept as holdback for morphological assessment. From the remaining wells 6 phenotypically normal embryos were collected and pooled in their vial groups into 1.5 mL Eppendorf tubes placed on ice 30 sec excess media removed placed back on ice 30 sec and 200 µL RNA shield added. Five pooled samples were collected from each treatment.	1mL of 1µm beads and 500µL of lysis buffer were loaded into a round bottom 2mL 96 well plate. 100µL of lysis buffer was added to tube containing pooled embryo samples and 200µL of RNA shield the full contents of the tube were then transferred to the homogenization plate. The plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 275 µL of the supernatant was then transferred to two plates containing 275µL of ethanol. RNA extraction was performed using the KingFisher Apex system. Briefly this process uses Mag beads to collect RNA from the sample post series of washes the RNA is eluted in 50µLof ultra pure water. RNA quality was assessed using the Agilent 4150 TapeStation System. All samples used had RIN scores >9.8. RNA concentration was determined using ThermoFisher Quant iT RNA Reagent and assay kit and ThermoFisher Varioskan ALF multimode plate reader in triplicate samples with high variability between reads were repeated in triplicate. The average of the three quantification reads was used for RNA concentration normalization. Each treatment had 4 5 viable RNA extraction replicates for treatments with 5 viable samples the 4 with the highest concentration and RIN score were used 3 control samples were used from each day group. All 94 samples were normalized to the lowest concentration sample 14.9ng/µL. 1 µL per sample of normalized RNA was used as input for library preparation following manufactures protocol for the 96 sample TempO Seq S1500 + Zebrafish Surrogate Assay panel BioSpyder Technologies Inc. Carlsbad CA United States. Briefly samples were added to the assay plate with an equal volume of 2x enhanced lysis buffer and processed through annealing with detector oligos digestion and ligation. Proper amplification was verified using real time PCR green fluorescence. Library purification was performed using the Macherey Nagel NucleoSpin Gel and PCR Cleanup Kit following adjustments detailed in the manufacturer’s instructions. PCR amplification products were pooled into a s…		tissue:whole animal\|genotype:5D WT\|treatment:1 ethylnaphthalene\|batch:1	GSM8969923	GSM8969923: 1 ethylnaphthalene B; Danio rerio; OTHER	GSM8969923 r1	GSM8969923	1	1mL of 1µm beads and 500µL of lysis buffer were loaded into a round bottom 2mL 96 well plate. 100µL of lysis buffer was added to tube containing pooled embryo samples and 200µL of RNA shield the full contents of the tube were then transferred to the homogenization plate. The plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 275 µL of the supernatant was then transferred to two plates containing 275µL of ethanol. RNA extraction was performed using the KingFisher Apex system. Briefly this process uses Mag beads to collect RNA from the sample post series of washes the RNA is eluted in 50µLof ultra pure water. RNA quality was assessed using the Agilent 4150 TapeStation System. All samples used had RIN scores >9.8. RNA concentration was determined using ThermoFisher Quant iT RNA Reagent and assay kit and ThermoFisher Varioskan ALF multimode plate reader in triplicate samples with high variability between reads were repeated in triplicate. The average of the three quantification reads was used for RNA concentration normalization. Each treatment had 4 5 viable RNA extraction replicates for treatments with 5 viable samples the 4 with the highest concentration and RIN score were used 3 control samples were used from each day group. All 94 samples were normalized to the lowest concentration sample 14.9ng/µL. 1 µL per sample of normalized RNA was used as input for library preparation following manufactures protocol for the 96 sample TempO Seq S1500 + Zebrafish Surrogate Assay panel BioSpyder Technologies Inc. Carlsbad CA United States. Briefly samples were added to the assay plate with an equal volume of 2x enhanced lysis buffer and processed through annealing with detector oligos digestion and ligation. Proper amplification was verified using real time PCR green fluorescence. Library purification was performed using the Macherey Nagel NucleoSpin Gel and PCR Cleanup Kit following adjustments detailed in the manufacturer's instructions. PCR amplification products were pooled into a s…	OTHER	TRANSCRIPTOMIC	other	SINGLE	ILLUMINA	Illumina NovaSeq X	SRP583274			8_B.fastq.gz	fastq	748961214.0	14685514.0	GSM8969923 r1	0:51	A:153969741;C:195524281;G:173004091;T:226292712;N:170389	51				153969741	195524281	173004091	226292712	170389	SRX28685516	SRS24952418	SRA2124446	SARL, Environmental and Molecular Toxicology, Oregon State University	SARL, Environmental and Molecular Toxicology, Oregon State University												B		usable mapping rate	illumina	novaseq_era	unknown	other	unknown	bulk	unknown	unknown	United States	2025-05-06	Multi-stage	Embryo	Whole Organism	All anatomical structures
36067	36067	SRR33444779	SRX28685515	SRS24952419	SRP583274	PRJNA1259243	Structure Dependent Developmental Toxicity of Alkyl Substituted Naphthalenes	GSE296410	Transcriptome Analysis	Naphthalene and its alkyl substituted derivatives are among the most abundant polycyclic aromatic hydrocarbons PAHs in environmental and human exposure studies yet their developmental toxicity and mode of action remain poorly understood due to challenges in testing of semi volatile compounds. This study developed a vial based high through put method to effectively assess the activity of naphthalenes and a set of 24 alkyl substituted naphthalenes. Early life stage zebrafish were exposed to a concentration series of each chemical 0 50 µM in rotating sealed glass vials to minimize volatilization. Benchmark concentration BMC50 values were calculated for morphological endpoints and lowest effect levels were determined for behavioral effects. The data were assessed for evidence of a narcotic mode of action using body burden measurements for select chemicals and logKow modeling. Targeted transcriptomics at a single concentration and timepoint as well as in silico molecular docking were conducted to generate mode of action hypothesis. The vial method enabled detection of highly variable developmental toxicity not previously observed using standard 96 well plate exposures. LogKow and body burden were poor predictors of toxicity suggesting a non narcotic mode of action. Transcriptomic analysis revealed a limited but notable set of differentially expressed genes with evidence for the disruption of glucocorticoid signaling pathways. Molecular docking identified potential protein targets e.g. CYP1A2 NT5E FOLR1 that may mediate observed effects. This study demonstrates the importance of appropriate exposure methods for semi volatile compounds reveals structure dependent toxicity among alkyl substituted naphthalenes and provides a foundation for further mechanistic studies and improved risk assessment of alkyl substituted PAHs. Overall design: Zebrafish were exposed to 25 chemicals naphthalene and 24 alkyl sustituted naphthalenes using a vial exposure method. Embryos were exposed to 20 uM of each chemical in groups of…				1 ethylnaphthalene A	GSM8969922		source name:whole animal\|tissue:whole animal\|genotype:5D WT\|treatment:1 ethylnaphthalene\|batch:1\|geo loc name:missing\|collection date:missing	1 ethylnaphthalene A	Demultiplexing and alignment was performed using the manufacture provided processing platform TempO SeqR BioSpyder Technologies Inc. Carlsbad CA United States. Counts of genes with multiple probes were summed as instructed by the manufacturer resulting in 3112 unique genes Read counts were normalized by upper quartile normalization using the calcNormFactors function from the Bioconductor package in EdgeR using the 75th percentile of reads per samples as the scaling factor. Principal component analysis PCA of normalized read counts was used to determine that a batch effect was present based on exposure and RNA extraction day nested by experimental design. At this time one control sample control day1 B was identified as an outlier and removed from all future analysis. The batch effect was removed from unnormalized data using the ComBat seq function from the SVA R package Normalzied read counts were normalized by upper quartile normalization using the calcNormFactors function from the Bioconductor package in EdgeR using the 75th percentile of reads per samples as the scaling factor. Assembly: GRCz11 Supplementary files format and content: .csv file containing raw count data Supplementary files format and content: .csv file containing batch corrected and normalized data	whole animal	When the embryos were 6hpf they were bleached in groups of 1000 or less. Embryos were placed in a strainer for transfer between solutions in 500 mL containers. First embryos were placed in system fish water for 1 minute then transferred to a 0.5% sodium chloride solution for 5 minutes then transferred to a % sodium hypochlorite solution for 1 minute and rinsed in EM for 5 minutes and transferred to a clean glass petri dish. Embryos were then loaded into 2mL glass exposure vials in groups of 8 using a glass pasture pipette. Excess EM was removed and 2mL of fresh EM was immediately added. Chemical stocks were diluted to 200 times the desired exposure concentration of 20 µM using DMSO. 10 µL of diluted chemical stock was added to each vial vials were immediately capped and inverted 3 times to ensure mixing no precipitate was observed for any chemicals at the concentrations tested. Vials were loaded into a custom 3D printed vial holder for slow end over end rotation in a dark 28°C incubator overnight. At 24 hours embryos were removed from exposure vials and plated into 96 well plates loaded with 100 µL clean EM. At 48 hpf embryos were collected for RNA extraction embryos from two exposure vials were kept as holdback for morphological assessment. From the remaining wells 6 phenotypically normal embryos were collected and pooled in their vial groups into 1.5 mL Eppendorf tubes placed on ice 30 sec excess media removed placed back on ice 30 sec and 200 µL RNA shield added. Five pooled samples were collected from each treatment.	1mL of 1µm beads and 500µL of lysis buffer were loaded into a round bottom 2mL 96 well plate. 100µL of lysis buffer was added to tube containing pooled embryo samples and 200µL of RNA shield the full contents of the tube were then transferred to the homogenization plate. The plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 275 µL of the supernatant was then transferred to two plates containing 275µL of ethanol. RNA extraction was performed using the KingFisher Apex system. Briefly this process uses Mag beads to collect RNA from the sample post series of washes the RNA is eluted in 50µLof ultra pure water. RNA quality was assessed using the Agilent 4150 TapeStation System. All samples used had RIN scores >9.8. RNA concentration was determined using ThermoFisher Quant iT RNA Reagent and assay kit and ThermoFisher Varioskan ALF multimode plate reader in triplicate samples with high variability between reads were repeated in triplicate. The average of the three quantification reads was used for RNA concentration normalization. Each treatment had 4 5 viable RNA extraction replicates for treatments with 5 viable samples the 4 with the highest concentration and RIN score were used 3 control samples were used from each day group. All 94 samples were normalized to the lowest concentration sample 14.9ng/µL. 1 µL per sample of normalized RNA was used as input for library preparation following manufactures protocol for the 96 sample TempO Seq S1500 + Zebrafish Surrogate Assay panel BioSpyder Technologies Inc. Carlsbad CA United States. Briefly samples were added to the assay plate with an equal volume of 2x enhanced lysis buffer and processed through annealing with detector oligos digestion and ligation. Proper amplification was verified using real time PCR green fluorescence. Library purification was performed using the Macherey Nagel NucleoSpin Gel and PCR Cleanup Kit following adjustments detailed in the manufacturer’s instructions. PCR amplification products were pooled into a s…		tissue:whole animal\|genotype:5D WT\|treatment:1 ethylnaphthalene\|batch:1	GSM8969922	GSM8969922: 1 ethylnaphthalene A; Danio rerio; OTHER	GSM8969922 r1	GSM8969922	1	1mL of 1µm beads and 500µL of lysis buffer were loaded into a round bottom 2mL 96 well plate. 100µL of lysis buffer was added to tube containing pooled embryo samples and 200µL of RNA shield the full contents of the tube were then transferred to the homogenization plate. The plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 275 µL of the supernatant was then transferred to two plates containing 275µL of ethanol. RNA extraction was performed using the KingFisher Apex system. Briefly this process uses Mag beads to collect RNA from the sample post series of washes the RNA is eluted in 50µLof ultra pure water. RNA quality was assessed using the Agilent 4150 TapeStation System. All samples used had RIN scores >9.8. RNA concentration was determined using ThermoFisher Quant iT RNA Reagent and assay kit and ThermoFisher Varioskan ALF multimode plate reader in triplicate samples with high variability between reads were repeated in triplicate. The average of the three quantification reads was used for RNA concentration normalization. Each treatment had 4 5 viable RNA extraction replicates for treatments with 5 viable samples the 4 with the highest concentration and RIN score were used 3 control samples were used from each day group. All 94 samples were normalized to the lowest concentration sample 14.9ng/µL. 1 µL per sample of normalized RNA was used as input for library preparation following manufactures protocol for the 96 sample TempO Seq S1500 + Zebrafish Surrogate Assay panel BioSpyder Technologies Inc. Carlsbad CA United States. Briefly samples were added to the assay plate with an equal volume of 2x enhanced lysis buffer and processed through annealing with detector oligos digestion and ligation. Proper amplification was verified using real time PCR green fluorescence. Library purification was performed using the Macherey Nagel NucleoSpin Gel and PCR Cleanup Kit following adjustments detailed in the manufacturer's instructions. PCR amplification products were pooled into a s…	OTHER	TRANSCRIPTOMIC	other	SINGLE	ILLUMINA	Illumina NovaSeq X	SRP583274			8_A.fastq.gz	fastq	587085225.0	11511475.0	GSM8969922 r1	0:51	A:119552515;C:153868067;G:135452522;T:178079346;N:132775	51				119552515	153868067	135452522	178079346	132775	SRX28685515	SRS24952419	SRA2124446	SARL, Environmental and Molecular Toxicology, Oregon State University	SARL, Environmental and Molecular Toxicology, Oregon State University												B		usable mapping rate	illumina	novaseq_era	unknown	other	unknown	bulk	unknown	unknown	United States	2025-05-06	Multi-stage	Embryo	Whole Organism	All anatomical structures
36068	36068	SRR33444780	SRX28685514	SRS24952417	SRP583274	PRJNA1259243	Structure Dependent Developmental Toxicity of Alkyl Substituted Naphthalenes	GSE296410	Transcriptome Analysis	Naphthalene and its alkyl substituted derivatives are among the most abundant polycyclic aromatic hydrocarbons PAHs in environmental and human exposure studies yet their developmental toxicity and mode of action remain poorly understood due to challenges in testing of semi volatile compounds. This study developed a vial based high through put method to effectively assess the activity of naphthalenes and a set of 24 alkyl substituted naphthalenes. Early life stage zebrafish were exposed to a concentration series of each chemical 0 50 µM in rotating sealed glass vials to minimize volatilization. Benchmark concentration BMC50 values were calculated for morphological endpoints and lowest effect levels were determined for behavioral effects. The data were assessed for evidence of a narcotic mode of action using body burden measurements for select chemicals and logKow modeling. Targeted transcriptomics at a single concentration and timepoint as well as in silico molecular docking were conducted to generate mode of action hypothesis. The vial method enabled detection of highly variable developmental toxicity not previously observed using standard 96 well plate exposures. LogKow and body burden were poor predictors of toxicity suggesting a non narcotic mode of action. Transcriptomic analysis revealed a limited but notable set of differentially expressed genes with evidence for the disruption of glucocorticoid signaling pathways. Molecular docking identified potential protein targets e.g. CYP1A2 NT5E FOLR1 that may mediate observed effects. This study demonstrates the importance of appropriate exposure methods for semi volatile compounds reveals structure dependent toxicity among alkyl substituted naphthalenes and provides a foundation for further mechanistic studies and improved risk assessment of alkyl substituted PAHs. Overall design: Zebrafish were exposed to 25 chemicals naphthalene and 24 alkyl sustituted naphthalenes using a vial exposure method. Embryos were exposed to 20 uM of each chemical in groups of…				naphthalene D	GSM8969889		source name:whole animal\|tissue:whole animal\|genotype:5D WT\|treatment:naphthalene\|batch:2\|geo loc name:missing\|collection date:missing	naphthalene D	Demultiplexing and alignment was performed using the manufacture provided processing platform TempO SeqR BioSpyder Technologies Inc. Carlsbad CA United States. Counts of genes with multiple probes were summed as instructed by the manufacturer resulting in 3112 unique genes Read counts were normalized by upper quartile normalization using the calcNormFactors function from the Bioconductor package in EdgeR using the 75th percentile of reads per samples as the scaling factor. Principal component analysis PCA of normalized read counts was used to determine that a batch effect was present based on exposure and RNA extraction day nested by experimental design. At this time one control sample control day1 B was identified as an outlier and removed from all future analysis. The batch effect was removed from unnormalized data using the ComBat seq function from the SVA R package Normalzied read counts were normalized by upper quartile normalization using the calcNormFactors function from the Bioconductor package in EdgeR using the 75th percentile of reads per samples as the scaling factor. Assembly: GRCz11 Supplementary files format and content: .csv file containing raw count data Supplementary files format and content: .csv file containing batch corrected and normalized data	whole animal	When the embryos were 6hpf they were bleached in groups of 1000 or less. Embryos were placed in a strainer for transfer between solutions in 500 mL containers. First embryos were placed in system fish water for 1 minute then transferred to a 0.5% sodium chloride solution for 5 minutes then transferred to a % sodium hypochlorite solution for 1 minute and rinsed in EM for 5 minutes and transferred to a clean glass petri dish. Embryos were then loaded into 2mL glass exposure vials in groups of 8 using a glass pasture pipette. Excess EM was removed and 2mL of fresh EM was immediately added. Chemical stocks were diluted to 200 times the desired exposure concentration of 20 µM using DMSO. 10 µL of diluted chemical stock was added to each vial vials were immediately capped and inverted 3 times to ensure mixing no precipitate was observed for any chemicals at the concentrations tested. Vials were loaded into a custom 3D printed vial holder for slow end over end rotation in a dark 28°C incubator overnight. At 24 hours embryos were removed from exposure vials and plated into 96 well plates loaded with 100 µL clean EM. At 48 hpf embryos were collected for RNA extraction embryos from two exposure vials were kept as holdback for morphological assessment. From the remaining wells 6 phenotypically normal embryos were collected and pooled in their vial groups into 1.5 mL Eppendorf tubes placed on ice 30 sec excess media removed placed back on ice 30 sec and 200 µL RNA shield added. Five pooled samples were collected from each treatment.	1mL of 1µm beads and 500µL of lysis buffer were loaded into a round bottom 2mL 96 well plate. 100µL of lysis buffer was added to tube containing pooled embryo samples and 200µL of RNA shield the full contents of the tube were then transferred to the homogenization plate. The plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 275 µL of the supernatant was then transferred to two plates containing 275µL of ethanol. RNA extraction was performed using the KingFisher Apex system. Briefly this process uses Mag beads to collect RNA from the sample post series of washes the RNA is eluted in 50µLof ultra pure water. RNA quality was assessed using the Agilent 4150 TapeStation System. All samples used had RIN scores >9.8. RNA concentration was determined using ThermoFisher Quant iT RNA Reagent and assay kit and ThermoFisher Varioskan ALF multimode plate reader in triplicate samples with high variability between reads were repeated in triplicate. The average of the three quantification reads was used for RNA concentration normalization. Each treatment had 4 5 viable RNA extraction replicates for treatments with 5 viable samples the 4 with the highest concentration and RIN score were used 3 control samples were used from each day group. All 94 samples were normalized to the lowest concentration sample 14.9ng/µL. 1 µL per sample of normalized RNA was used as input for library preparation following manufactures protocol for the 96 sample TempO Seq S1500 + Zebrafish Surrogate Assay panel BioSpyder Technologies Inc. Carlsbad CA United States. Briefly samples were added to the assay plate with an equal volume of 2x enhanced lysis buffer and processed through annealing with detector oligos digestion and ligation. Proper amplification was verified using real time PCR green fluorescence. Library purification was performed using the Macherey Nagel NucleoSpin Gel and PCR Cleanup Kit following adjustments detailed in the manufacturer’s instructions. PCR amplification products were pooled into a s…		tissue:whole animal\|genotype:5D WT\|treatment:naphthalene\|batch:2	GSM8969889	GSM8969889: naphthalene D; Danio rerio; OTHER	GSM8969889 r1	GSM8969889	1	1mL of 1µm beads and 500µL of lysis buffer were loaded into a round bottom 2mL 96 well plate. 100µL of lysis buffer was added to tube containing pooled embryo samples and 200µL of RNA shield the full contents of the tube were then transferred to the homogenization plate. The plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 275 µL of the supernatant was then transferred to two plates containing 275µL of ethanol. RNA extraction was performed using the KingFisher Apex system. Briefly this process uses Mag beads to collect RNA from the sample post series of washes the RNA is eluted in 50µLof ultra pure water. RNA quality was assessed using the Agilent 4150 TapeStation System. All samples used had RIN scores >9.8. RNA concentration was determined using ThermoFisher Quant iT RNA Reagent and assay kit and ThermoFisher Varioskan ALF multimode plate reader in triplicate samples with high variability between reads were repeated in triplicate. The average of the three quantification reads was used for RNA concentration normalization. Each treatment had 4 5 viable RNA extraction replicates for treatments with 5 viable samples the 4 with the highest concentration and RIN score were used 3 control samples were used from each day group. All 94 samples were normalized to the lowest concentration sample 14.9ng/µL. 1 µL per sample of normalized RNA was used as input for library preparation following manufactures protocol for the 96 sample TempO Seq S1500 + Zebrafish Surrogate Assay panel BioSpyder Technologies Inc. Carlsbad CA United States. Briefly samples were added to the assay plate with an equal volume of 2x enhanced lysis buffer and processed through annealing with detector oligos digestion and ligation. Proper amplification was verified using real time PCR green fluorescence. Library purification was performed using the Macherey Nagel NucleoSpin Gel and PCR Cleanup Kit following adjustments detailed in the manufacturer's instructions. PCR amplification products were pooled into a s…	OTHER	TRANSCRIPTOMIC	other	SINGLE	ILLUMINA	Illumina NovaSeq X	SRP583274			20D.fastq.gz	fastq	574442223.0	11263573.0	GSM8969889 r1	0:51	A:115679820;C:152904111;G:134156914;T:171570534;N:130844	51				115679820	152904111	134156914	171570534	130844	SRX28685514	SRS24952417	SRA2124446	SARL, Environmental and Molecular Toxicology, Oregon State University	SARL, Environmental and Molecular Toxicology, Oregon State University												B		usable mapping rate	illumina	novaseq_era	unknown	other	unknown	bulk	unknown	unknown	United States	2025-05-06	Multi-stage	Embryo	Whole Organism	All anatomical structures
36069	36069	SRR33444781	SRX28685513	SRS24952416	SRP583274	PRJNA1259243	Structure Dependent Developmental Toxicity of Alkyl Substituted Naphthalenes	GSE296410	Transcriptome Analysis	Naphthalene and its alkyl substituted derivatives are among the most abundant polycyclic aromatic hydrocarbons PAHs in environmental and human exposure studies yet their developmental toxicity and mode of action remain poorly understood due to challenges in testing of semi volatile compounds. This study developed a vial based high through put method to effectively assess the activity of naphthalenes and a set of 24 alkyl substituted naphthalenes. Early life stage zebrafish were exposed to a concentration series of each chemical 0 50 µM in rotating sealed glass vials to minimize volatilization. Benchmark concentration BMC50 values were calculated for morphological endpoints and lowest effect levels were determined for behavioral effects. The data were assessed for evidence of a narcotic mode of action using body burden measurements for select chemicals and logKow modeling. Targeted transcriptomics at a single concentration and timepoint as well as in silico molecular docking were conducted to generate mode of action hypothesis. The vial method enabled detection of highly variable developmental toxicity not previously observed using standard 96 well plate exposures. LogKow and body burden were poor predictors of toxicity suggesting a non narcotic mode of action. Transcriptomic analysis revealed a limited but notable set of differentially expressed genes with evidence for the disruption of glucocorticoid signaling pathways. Molecular docking identified potential protein targets e.g. CYP1A2 NT5E FOLR1 that may mediate observed effects. This study demonstrates the importance of appropriate exposure methods for semi volatile compounds reveals structure dependent toxicity among alkyl substituted naphthalenes and provides a foundation for further mechanistic studies and improved risk assessment of alkyl substituted PAHs. Overall design: Zebrafish were exposed to 25 chemicals naphthalene and 24 alkyl sustituted naphthalenes using a vial exposure method. Embryos were exposed to 20 uM of each chemical in groups of…				naphthalene C	GSM8969888		source name:whole animal\|tissue:whole animal\|genotype:5D WT\|treatment:naphthalene\|batch:2\|geo loc name:missing\|collection date:missing	naphthalene C	Demultiplexing and alignment was performed using the manufacture provided processing platform TempO SeqR BioSpyder Technologies Inc. Carlsbad CA United States. Counts of genes with multiple probes were summed as instructed by the manufacturer resulting in 3112 unique genes Read counts were normalized by upper quartile normalization using the calcNormFactors function from the Bioconductor package in EdgeR using the 75th percentile of reads per samples as the scaling factor. Principal component analysis PCA of normalized read counts was used to determine that a batch effect was present based on exposure and RNA extraction day nested by experimental design. At this time one control sample control day1 B was identified as an outlier and removed from all future analysis. The batch effect was removed from unnormalized data using the ComBat seq function from the SVA R package Normalzied read counts were normalized by upper quartile normalization using the calcNormFactors function from the Bioconductor package in EdgeR using the 75th percentile of reads per samples as the scaling factor. Assembly: GRCz11 Supplementary files format and content: .csv file containing raw count data Supplementary files format and content: .csv file containing batch corrected and normalized data	whole animal	When the embryos were 6hpf they were bleached in groups of 1000 or less. Embryos were placed in a strainer for transfer between solutions in 500 mL containers. First embryos were placed in system fish water for 1 minute then transferred to a 0.5% sodium chloride solution for 5 minutes then transferred to a % sodium hypochlorite solution for 1 minute and rinsed in EM for 5 minutes and transferred to a clean glass petri dish. Embryos were then loaded into 2mL glass exposure vials in groups of 8 using a glass pasture pipette. Excess EM was removed and 2mL of fresh EM was immediately added. Chemical stocks were diluted to 200 times the desired exposure concentration of 20 µM using DMSO. 10 µL of diluted chemical stock was added to each vial vials were immediately capped and inverted 3 times to ensure mixing no precipitate was observed for any chemicals at the concentrations tested. Vials were loaded into a custom 3D printed vial holder for slow end over end rotation in a dark 28°C incubator overnight. At 24 hours embryos were removed from exposure vials and plated into 96 well plates loaded with 100 µL clean EM. At 48 hpf embryos were collected for RNA extraction embryos from two exposure vials were kept as holdback for morphological assessment. From the remaining wells 6 phenotypically normal embryos were collected and pooled in their vial groups into 1.5 mL Eppendorf tubes placed on ice 30 sec excess media removed placed back on ice 30 sec and 200 µL RNA shield added. Five pooled samples were collected from each treatment.	1mL of 1µm beads and 500µL of lysis buffer were loaded into a round bottom 2mL 96 well plate. 100µL of lysis buffer was added to tube containing pooled embryo samples and 200µL of RNA shield the full contents of the tube were then transferred to the homogenization plate. The plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 275 µL of the supernatant was then transferred to two plates containing 275µL of ethanol. RNA extraction was performed using the KingFisher Apex system. Briefly this process uses Mag beads to collect RNA from the sample post series of washes the RNA is eluted in 50µLof ultra pure water. RNA quality was assessed using the Agilent 4150 TapeStation System. All samples used had RIN scores >9.8. RNA concentration was determined using ThermoFisher Quant iT RNA Reagent and assay kit and ThermoFisher Varioskan ALF multimode plate reader in triplicate samples with high variability between reads were repeated in triplicate. The average of the three quantification reads was used for RNA concentration normalization. Each treatment had 4 5 viable RNA extraction replicates for treatments with 5 viable samples the 4 with the highest concentration and RIN score were used 3 control samples were used from each day group. All 94 samples were normalized to the lowest concentration sample 14.9ng/µL. 1 µL per sample of normalized RNA was used as input for library preparation following manufactures protocol for the 96 sample TempO Seq S1500 + Zebrafish Surrogate Assay panel BioSpyder Technologies Inc. Carlsbad CA United States. Briefly samples were added to the assay plate with an equal volume of 2x enhanced lysis buffer and processed through annealing with detector oligos digestion and ligation. Proper amplification was verified using real time PCR green fluorescence. Library purification was performed using the Macherey Nagel NucleoSpin Gel and PCR Cleanup Kit following adjustments detailed in the manufacturer’s instructions. PCR amplification products were pooled into a s…		tissue:whole animal\|genotype:5D WT\|treatment:naphthalene\|batch:2	GSM8969888	GSM8969888: naphthalene C; Danio rerio; OTHER	GSM8969888 r1	GSM8969888	1	1mL of 1µm beads and 500µL of lysis buffer were loaded into a round bottom 2mL 96 well plate. 100µL of lysis buffer was added to tube containing pooled embryo samples and 200µL of RNA shield the full contents of the tube were then transferred to the homogenization plate. The plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 275 µL of the supernatant was then transferred to two plates containing 275µL of ethanol. RNA extraction was performed using the KingFisher Apex system. Briefly this process uses Mag beads to collect RNA from the sample post series of washes the RNA is eluted in 50µLof ultra pure water. RNA quality was assessed using the Agilent 4150 TapeStation System. All samples used had RIN scores >9.8. RNA concentration was determined using ThermoFisher Quant iT RNA Reagent and assay kit and ThermoFisher Varioskan ALF multimode plate reader in triplicate samples with high variability between reads were repeated in triplicate. The average of the three quantification reads was used for RNA concentration normalization. Each treatment had 4 5 viable RNA extraction replicates for treatments with 5 viable samples the 4 with the highest concentration and RIN score were used 3 control samples were used from each day group. All 94 samples were normalized to the lowest concentration sample 14.9ng/µL. 1 µL per sample of normalized RNA was used as input for library preparation following manufactures protocol for the 96 sample TempO Seq S1500 + Zebrafish Surrogate Assay panel BioSpyder Technologies Inc. Carlsbad CA United States. Briefly samples were added to the assay plate with an equal volume of 2x enhanced lysis buffer and processed through annealing with detector oligos digestion and ligation. Proper amplification was verified using real time PCR green fluorescence. Library purification was performed using the Macherey Nagel NucleoSpin Gel and PCR Cleanup Kit following adjustments detailed in the manufacturer's instructions. PCR amplification products were pooled into a s…	OTHER	TRANSCRIPTOMIC	other	SINGLE	ILLUMINA	Illumina NovaSeq X	SRP583274			20C.fastq.gz	fastq	626063199.0	12275749.0	GSM8969888 r1	0:51	A:126797180;C:165522830;G:146080993;T:187520712;N:141484	51				126797180	165522830	146080993	187520712	141484	SRX28685513	SRS24952416	SRA2124446	SARL, Environmental and Molecular Toxicology, Oregon State University	SARL, Environmental and Molecular Toxicology, Oregon State University												B		usable mapping rate	illumina	novaseq_era	unknown	other	unknown	bulk	unknown	unknown	United States	2025-05-06	Multi-stage	Embryo	Whole Organism	All anatomical structures
36070	36070	SRR33444782	SRX28685512	SRS24952415	SRP583274	PRJNA1259243	Structure Dependent Developmental Toxicity of Alkyl Substituted Naphthalenes	GSE296410	Transcriptome Analysis	Naphthalene and its alkyl substituted derivatives are among the most abundant polycyclic aromatic hydrocarbons PAHs in environmental and human exposure studies yet their developmental toxicity and mode of action remain poorly understood due to challenges in testing of semi volatile compounds. This study developed a vial based high through put method to effectively assess the activity of naphthalenes and a set of 24 alkyl substituted naphthalenes. Early life stage zebrafish were exposed to a concentration series of each chemical 0 50 µM in rotating sealed glass vials to minimize volatilization. Benchmark concentration BMC50 values were calculated for morphological endpoints and lowest effect levels were determined for behavioral effects. The data were assessed for evidence of a narcotic mode of action using body burden measurements for select chemicals and logKow modeling. Targeted transcriptomics at a single concentration and timepoint as well as in silico molecular docking were conducted to generate mode of action hypothesis. The vial method enabled detection of highly variable developmental toxicity not previously observed using standard 96 well plate exposures. LogKow and body burden were poor predictors of toxicity suggesting a non narcotic mode of action. Transcriptomic analysis revealed a limited but notable set of differentially expressed genes with evidence for the disruption of glucocorticoid signaling pathways. Molecular docking identified potential protein targets e.g. CYP1A2 NT5E FOLR1 that may mediate observed effects. This study demonstrates the importance of appropriate exposure methods for semi volatile compounds reveals structure dependent toxicity among alkyl substituted naphthalenes and provides a foundation for further mechanistic studies and improved risk assessment of alkyl substituted PAHs. Overall design: Zebrafish were exposed to 25 chemicals naphthalene and 24 alkyl sustituted naphthalenes using a vial exposure method. Embryos were exposed to 20 uM of each chemical in groups of…				naphthalene B	GSM8969887		source name:whole animal\|tissue:whole animal\|genotype:5D WT\|treatment:naphthalene\|batch:2\|geo loc name:missing\|collection date:missing	naphthalene B	Demultiplexing and alignment was performed using the manufacture provided processing platform TempO SeqR BioSpyder Technologies Inc. Carlsbad CA United States. Counts of genes with multiple probes were summed as instructed by the manufacturer resulting in 3112 unique genes Read counts were normalized by upper quartile normalization using the calcNormFactors function from the Bioconductor package in EdgeR using the 75th percentile of reads per samples as the scaling factor. Principal component analysis PCA of normalized read counts was used to determine that a batch effect was present based on exposure and RNA extraction day nested by experimental design. At this time one control sample control day1 B was identified as an outlier and removed from all future analysis. The batch effect was removed from unnormalized data using the ComBat seq function from the SVA R package Normalzied read counts were normalized by upper quartile normalization using the calcNormFactors function from the Bioconductor package in EdgeR using the 75th percentile of reads per samples as the scaling factor. Assembly: GRCz11 Supplementary files format and content: .csv file containing raw count data Supplementary files format and content: .csv file containing batch corrected and normalized data	whole animal	When the embryos were 6hpf they were bleached in groups of 1000 or less. Embryos were placed in a strainer for transfer between solutions in 500 mL containers. First embryos were placed in system fish water for 1 minute then transferred to a 0.5% sodium chloride solution for 5 minutes then transferred to a % sodium hypochlorite solution for 1 minute and rinsed in EM for 5 minutes and transferred to a clean glass petri dish. Embryos were then loaded into 2mL glass exposure vials in groups of 8 using a glass pasture pipette. Excess EM was removed and 2mL of fresh EM was immediately added. Chemical stocks were diluted to 200 times the desired exposure concentration of 20 µM using DMSO. 10 µL of diluted chemical stock was added to each vial vials were immediately capped and inverted 3 times to ensure mixing no precipitate was observed for any chemicals at the concentrations tested. Vials were loaded into a custom 3D printed vial holder for slow end over end rotation in a dark 28°C incubator overnight. At 24 hours embryos were removed from exposure vials and plated into 96 well plates loaded with 100 µL clean EM. At 48 hpf embryos were collected for RNA extraction embryos from two exposure vials were kept as holdback for morphological assessment. From the remaining wells 6 phenotypically normal embryos were collected and pooled in their vial groups into 1.5 mL Eppendorf tubes placed on ice 30 sec excess media removed placed back on ice 30 sec and 200 µL RNA shield added. Five pooled samples were collected from each treatment.	1mL of 1µm beads and 500µL of lysis buffer were loaded into a round bottom 2mL 96 well plate. 100µL of lysis buffer was added to tube containing pooled embryo samples and 200µL of RNA shield the full contents of the tube were then transferred to the homogenization plate. The plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 275 µL of the supernatant was then transferred to two plates containing 275µL of ethanol. RNA extraction was performed using the KingFisher Apex system. Briefly this process uses Mag beads to collect RNA from the sample post series of washes the RNA is eluted in 50µLof ultra pure water. RNA quality was assessed using the Agilent 4150 TapeStation System. All samples used had RIN scores >9.8. RNA concentration was determined using ThermoFisher Quant iT RNA Reagent and assay kit and ThermoFisher Varioskan ALF multimode plate reader in triplicate samples with high variability between reads were repeated in triplicate. The average of the three quantification reads was used for RNA concentration normalization. Each treatment had 4 5 viable RNA extraction replicates for treatments with 5 viable samples the 4 with the highest concentration and RIN score were used 3 control samples were used from each day group. All 94 samples were normalized to the lowest concentration sample 14.9ng/µL. 1 µL per sample of normalized RNA was used as input for library preparation following manufactures protocol for the 96 sample TempO Seq S1500 + Zebrafish Surrogate Assay panel BioSpyder Technologies Inc. Carlsbad CA United States. Briefly samples were added to the assay plate with an equal volume of 2x enhanced lysis buffer and processed through annealing with detector oligos digestion and ligation. Proper amplification was verified using real time PCR green fluorescence. Library purification was performed using the Macherey Nagel NucleoSpin Gel and PCR Cleanup Kit following adjustments detailed in the manufacturer’s instructions. PCR amplification products were pooled into a s…		tissue:whole animal\|genotype:5D WT\|treatment:naphthalene\|batch:2	GSM8969887	GSM8969887: naphthalene B; Danio rerio; OTHER	GSM8969887 r1	GSM8969887	1	1mL of 1µm beads and 500µL of lysis buffer were loaded into a round bottom 2mL 96 well plate. 100µL of lysis buffer was added to tube containing pooled embryo samples and 200µL of RNA shield the full contents of the tube were then transferred to the homogenization plate. The plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 275 µL of the supernatant was then transferred to two plates containing 275µL of ethanol. RNA extraction was performed using the KingFisher Apex system. Briefly this process uses Mag beads to collect RNA from the sample post series of washes the RNA is eluted in 50µLof ultra pure water. RNA quality was assessed using the Agilent 4150 TapeStation System. All samples used had RIN scores >9.8. RNA concentration was determined using ThermoFisher Quant iT RNA Reagent and assay kit and ThermoFisher Varioskan ALF multimode plate reader in triplicate samples with high variability between reads were repeated in triplicate. The average of the three quantification reads was used for RNA concentration normalization. Each treatment had 4 5 viable RNA extraction replicates for treatments with 5 viable samples the 4 with the highest concentration and RIN score were used 3 control samples were used from each day group. All 94 samples were normalized to the lowest concentration sample 14.9ng/µL. 1 µL per sample of normalized RNA was used as input for library preparation following manufactures protocol for the 96 sample TempO Seq S1500 + Zebrafish Surrogate Assay panel BioSpyder Technologies Inc. Carlsbad CA United States. Briefly samples were added to the assay plate with an equal volume of 2x enhanced lysis buffer and processed through annealing with detector oligos digestion and ligation. Proper amplification was verified using real time PCR green fluorescence. Library purification was performed using the Macherey Nagel NucleoSpin Gel and PCR Cleanup Kit following adjustments detailed in the manufacturer's instructions. PCR amplification products were pooled into a s…	OTHER	TRANSCRIPTOMIC	other	SINGLE	ILLUMINA	Illumina NovaSeq X	SRP583274			20B.fastq.gz	fastq	743186535.0	14572285.0	GSM8969887 r1	0:51	A:149056375;C:196734696;G:173272512;T:223954929;N:168023	51				149056375	196734696	173272512	223954929	168023	SRX28685512	SRS24952415	SRA2124446	SARL, Environmental and Molecular Toxicology, Oregon State University	SARL, Environmental and Molecular Toxicology, Oregon State University												B		usable mapping rate	illumina	novaseq_era	unknown	other	unknown	bulk	unknown	unknown	United States	2025-05-06	Multi-stage	Embryo	Whole Organism	All anatomical structures
36071	36071	SRR33444783	SRX28685511	SRS24952413	SRP583274	PRJNA1259243	Structure Dependent Developmental Toxicity of Alkyl Substituted Naphthalenes	GSE296410	Transcriptome Analysis	Naphthalene and its alkyl substituted derivatives are among the most abundant polycyclic aromatic hydrocarbons PAHs in environmental and human exposure studies yet their developmental toxicity and mode of action remain poorly understood due to challenges in testing of semi volatile compounds. This study developed a vial based high through put method to effectively assess the activity of naphthalenes and a set of 24 alkyl substituted naphthalenes. Early life stage zebrafish were exposed to a concentration series of each chemical 0 50 µM in rotating sealed glass vials to minimize volatilization. Benchmark concentration BMC50 values were calculated for morphological endpoints and lowest effect levels were determined for behavioral effects. The data were assessed for evidence of a narcotic mode of action using body burden measurements for select chemicals and logKow modeling. Targeted transcriptomics at a single concentration and timepoint as well as in silico molecular docking were conducted to generate mode of action hypothesis. The vial method enabled detection of highly variable developmental toxicity not previously observed using standard 96 well plate exposures. LogKow and body burden were poor predictors of toxicity suggesting a non narcotic mode of action. Transcriptomic analysis revealed a limited but notable set of differentially expressed genes with evidence for the disruption of glucocorticoid signaling pathways. Molecular docking identified potential protein targets e.g. CYP1A2 NT5E FOLR1 that may mediate observed effects. This study demonstrates the importance of appropriate exposure methods for semi volatile compounds reveals structure dependent toxicity among alkyl substituted naphthalenes and provides a foundation for further mechanistic studies and improved risk assessment of alkyl substituted PAHs. Overall design: Zebrafish were exposed to 25 chemicals naphthalene and 24 alkyl sustituted naphthalenes using a vial exposure method. Embryos were exposed to 20 uM of each chemical in groups of…				naphthalene A	GSM8969886		source name:whole animal\|tissue:whole animal\|genotype:5D WT\|treatment:naphthalene\|batch:2\|geo loc name:missing\|collection date:missing	naphthalene A	Demultiplexing and alignment was performed using the manufacture provided processing platform TempO SeqR BioSpyder Technologies Inc. Carlsbad CA United States. Counts of genes with multiple probes were summed as instructed by the manufacturer resulting in 3112 unique genes Read counts were normalized by upper quartile normalization using the calcNormFactors function from the Bioconductor package in EdgeR using the 75th percentile of reads per samples as the scaling factor. Principal component analysis PCA of normalized read counts was used to determine that a batch effect was present based on exposure and RNA extraction day nested by experimental design. At this time one control sample control day1 B was identified as an outlier and removed from all future analysis. The batch effect was removed from unnormalized data using the ComBat seq function from the SVA R package Normalzied read counts were normalized by upper quartile normalization using the calcNormFactors function from the Bioconductor package in EdgeR using the 75th percentile of reads per samples as the scaling factor. Assembly: GRCz11 Supplementary files format and content: .csv file containing raw count data Supplementary files format and content: .csv file containing batch corrected and normalized data	whole animal	When the embryos were 6hpf they were bleached in groups of 1000 or less. Embryos were placed in a strainer for transfer between solutions in 500 mL containers. First embryos were placed in system fish water for 1 minute then transferred to a 0.5% sodium chloride solution for 5 minutes then transferred to a % sodium hypochlorite solution for 1 minute and rinsed in EM for 5 minutes and transferred to a clean glass petri dish. Embryos were then loaded into 2mL glass exposure vials in groups of 8 using a glass pasture pipette. Excess EM was removed and 2mL of fresh EM was immediately added. Chemical stocks were diluted to 200 times the desired exposure concentration of 20 µM using DMSO. 10 µL of diluted chemical stock was added to each vial vials were immediately capped and inverted 3 times to ensure mixing no precipitate was observed for any chemicals at the concentrations tested. Vials were loaded into a custom 3D printed vial holder for slow end over end rotation in a dark 28°C incubator overnight. At 24 hours embryos were removed from exposure vials and plated into 96 well plates loaded with 100 µL clean EM. At 48 hpf embryos were collected for RNA extraction embryos from two exposure vials were kept as holdback for morphological assessment. From the remaining wells 6 phenotypically normal embryos were collected and pooled in their vial groups into 1.5 mL Eppendorf tubes placed on ice 30 sec excess media removed placed back on ice 30 sec and 200 µL RNA shield added. Five pooled samples were collected from each treatment.	1mL of 1µm beads and 500µL of lysis buffer were loaded into a round bottom 2mL 96 well plate. 100µL of lysis buffer was added to tube containing pooled embryo samples and 200µL of RNA shield the full contents of the tube were then transferred to the homogenization plate. The plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 275 µL of the supernatant was then transferred to two plates containing 275µL of ethanol. RNA extraction was performed using the KingFisher Apex system. Briefly this process uses Mag beads to collect RNA from the sample post series of washes the RNA is eluted in 50µLof ultra pure water. RNA quality was assessed using the Agilent 4150 TapeStation System. All samples used had RIN scores >9.8. RNA concentration was determined using ThermoFisher Quant iT RNA Reagent and assay kit and ThermoFisher Varioskan ALF multimode plate reader in triplicate samples with high variability between reads were repeated in triplicate. The average of the three quantification reads was used for RNA concentration normalization. Each treatment had 4 5 viable RNA extraction replicates for treatments with 5 viable samples the 4 with the highest concentration and RIN score were used 3 control samples were used from each day group. All 94 samples were normalized to the lowest concentration sample 14.9ng/µL. 1 µL per sample of normalized RNA was used as input for library preparation following manufactures protocol for the 96 sample TempO Seq S1500 + Zebrafish Surrogate Assay panel BioSpyder Technologies Inc. Carlsbad CA United States. Briefly samples were added to the assay plate with an equal volume of 2x enhanced lysis buffer and processed through annealing with detector oligos digestion and ligation. Proper amplification was verified using real time PCR green fluorescence. Library purification was performed using the Macherey Nagel NucleoSpin Gel and PCR Cleanup Kit following adjustments detailed in the manufacturer’s instructions. PCR amplification products were pooled into a s…		tissue:whole animal\|genotype:5D WT\|treatment:naphthalene\|batch:2	GSM8969886	GSM8969886: naphthalene A; Danio rerio; OTHER	GSM8969886 r1	GSM8969886	1	1mL of 1µm beads and 500µL of lysis buffer were loaded into a round bottom 2mL 96 well plate. 100µL of lysis buffer was added to tube containing pooled embryo samples and 200µL of RNA shield the full contents of the tube were then transferred to the homogenization plate. The plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 275 µL of the supernatant was then transferred to two plates containing 275µL of ethanol. RNA extraction was performed using the KingFisher Apex system. Briefly this process uses Mag beads to collect RNA from the sample post series of washes the RNA is eluted in 50µLof ultra pure water. RNA quality was assessed using the Agilent 4150 TapeStation System. All samples used had RIN scores >9.8. RNA concentration was determined using ThermoFisher Quant iT RNA Reagent and assay kit and ThermoFisher Varioskan ALF multimode plate reader in triplicate samples with high variability between reads were repeated in triplicate. The average of the three quantification reads was used for RNA concentration normalization. Each treatment had 4 5 viable RNA extraction replicates for treatments with 5 viable samples the 4 with the highest concentration and RIN score were used 3 control samples were used from each day group. All 94 samples were normalized to the lowest concentration sample 14.9ng/µL. 1 µL per sample of normalized RNA was used as input for library preparation following manufactures protocol for the 96 sample TempO Seq S1500 + Zebrafish Surrogate Assay panel BioSpyder Technologies Inc. Carlsbad CA United States. Briefly samples were added to the assay plate with an equal volume of 2x enhanced lysis buffer and processed through annealing with detector oligos digestion and ligation. Proper amplification was verified using real time PCR green fluorescence. Library purification was performed using the Macherey Nagel NucleoSpin Gel and PCR Cleanup Kit following adjustments detailed in the manufacturer's instructions. PCR amplification products were pooled into a s…	OTHER	TRANSCRIPTOMIC	other	SINGLE	ILLUMINA	Illumina NovaSeq X	SRP583274			20A.fastq.gz	fastq	586121376.0	11492576.0	GSM8969886 r1	0:51	A:117510651;C:155746473;G:136839801;T:175891905;N:132546	51				117510651	155746473	136839801	175891905	132546	SRX28685511	SRS24952413	SRA2124446	SARL, Environmental and Molecular Toxicology, Oregon State University	SARL, Environmental and Molecular Toxicology, Oregon State University												B		usable mapping rate	illumina	novaseq_era	unknown	other	unknown	bulk	unknown	unknown	United States	2025-05-06	Multi-stage	Embryo	Whole Organism	All anatomical structures
36072	36072	SRR33444784	SRX28685510	SRS24952414	SRP583274	PRJNA1259243	Structure Dependent Developmental Toxicity of Alkyl Substituted Naphthalenes	GSE296410	Transcriptome Analysis	Naphthalene and its alkyl substituted derivatives are among the most abundant polycyclic aromatic hydrocarbons PAHs in environmental and human exposure studies yet their developmental toxicity and mode of action remain poorly understood due to challenges in testing of semi volatile compounds. This study developed a vial based high through put method to effectively assess the activity of naphthalenes and a set of 24 alkyl substituted naphthalenes. Early life stage zebrafish were exposed to a concentration series of each chemical 0 50 µM in rotating sealed glass vials to minimize volatilization. Benchmark concentration BMC50 values were calculated for morphological endpoints and lowest effect levels were determined for behavioral effects. The data were assessed for evidence of a narcotic mode of action using body burden measurements for select chemicals and logKow modeling. Targeted transcriptomics at a single concentration and timepoint as well as in silico molecular docking were conducted to generate mode of action hypothesis. The vial method enabled detection of highly variable developmental toxicity not previously observed using standard 96 well plate exposures. LogKow and body burden were poor predictors of toxicity suggesting a non narcotic mode of action. Transcriptomic analysis revealed a limited but notable set of differentially expressed genes with evidence for the disruption of glucocorticoid signaling pathways. Molecular docking identified potential protein targets e.g. CYP1A2 NT5E FOLR1 that may mediate observed effects. This study demonstrates the importance of appropriate exposure methods for semi volatile compounds reveals structure dependent toxicity among alkyl substituted naphthalenes and provides a foundation for further mechanistic studies and improved risk assessment of alkyl substituted PAHs. Overall design: Zebrafish were exposed to 25 chemicals naphthalene and 24 alkyl sustituted naphthalenes using a vial exposure method. Embryos were exposed to 20 uM of each chemical in groups of…				2 6 diethylnaphthalene E	GSM8969885		source name:whole animal\|tissue:whole animal\|genotype:5D WT\|treatment:2 6 diethylnaphthalene\|batch:1\|geo loc name:missing\|collection date:missing	2 6 diethylnaphthalene E	Demultiplexing and alignment was performed using the manufacture provided processing platform TempO SeqR BioSpyder Technologies Inc. Carlsbad CA United States. Counts of genes with multiple probes were summed as instructed by the manufacturer resulting in 3112 unique genes Read counts were normalized by upper quartile normalization using the calcNormFactors function from the Bioconductor package in EdgeR using the 75th percentile of reads per samples as the scaling factor. Principal component analysis PCA of normalized read counts was used to determine that a batch effect was present based on exposure and RNA extraction day nested by experimental design. At this time one control sample control day1 B was identified as an outlier and removed from all future analysis. The batch effect was removed from unnormalized data using the ComBat seq function from the SVA R package Normalzied read counts were normalized by upper quartile normalization using the calcNormFactors function from the Bioconductor package in EdgeR using the 75th percentile of reads per samples as the scaling factor. Assembly: GRCz11 Supplementary files format and content: .csv file containing raw count data Supplementary files format and content: .csv file containing batch corrected and normalized data	whole animal	When the embryos were 6hpf they were bleached in groups of 1000 or less. Embryos were placed in a strainer for transfer between solutions in 500 mL containers. First embryos were placed in system fish water for 1 minute then transferred to a 0.5% sodium chloride solution for 5 minutes then transferred to a % sodium hypochlorite solution for 1 minute and rinsed in EM for 5 minutes and transferred to a clean glass petri dish. Embryos were then loaded into 2mL glass exposure vials in groups of 8 using a glass pasture pipette. Excess EM was removed and 2mL of fresh EM was immediately added. Chemical stocks were diluted to 200 times the desired exposure concentration of 20 µM using DMSO. 10 µL of diluted chemical stock was added to each vial vials were immediately capped and inverted 3 times to ensure mixing no precipitate was observed for any chemicals at the concentrations tested. Vials were loaded into a custom 3D printed vial holder for slow end over end rotation in a dark 28°C incubator overnight. At 24 hours embryos were removed from exposure vials and plated into 96 well plates loaded with 100 µL clean EM. At 48 hpf embryos were collected for RNA extraction embryos from two exposure vials were kept as holdback for morphological assessment. From the remaining wells 6 phenotypically normal embryos were collected and pooled in their vial groups into 1.5 mL Eppendorf tubes placed on ice 30 sec excess media removed placed back on ice 30 sec and 200 µL RNA shield added. Five pooled samples were collected from each treatment.	1mL of 1µm beads and 500µL of lysis buffer were loaded into a round bottom 2mL 96 well plate. 100µL of lysis buffer was added to tube containing pooled embryo samples and 200µL of RNA shield the full contents of the tube were then transferred to the homogenization plate. The plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 275 µL of the supernatant was then transferred to two plates containing 275µL of ethanol. RNA extraction was performed using the KingFisher Apex system. Briefly this process uses Mag beads to collect RNA from the sample post series of washes the RNA is eluted in 50µLof ultra pure water. RNA quality was assessed using the Agilent 4150 TapeStation System. All samples used had RIN scores >9.8. RNA concentration was determined using ThermoFisher Quant iT RNA Reagent and assay kit and ThermoFisher Varioskan ALF multimode plate reader in triplicate samples with high variability between reads were repeated in triplicate. The average of the three quantification reads was used for RNA concentration normalization. Each treatment had 4 5 viable RNA extraction replicates for treatments with 5 viable samples the 4 with the highest concentration and RIN score were used 3 control samples were used from each day group. All 94 samples were normalized to the lowest concentration sample 14.9ng/µL. 1 µL per sample of normalized RNA was used as input for library preparation following manufactures protocol for the 96 sample TempO Seq S1500 + Zebrafish Surrogate Assay panel BioSpyder Technologies Inc. Carlsbad CA United States. Briefly samples were added to the assay plate with an equal volume of 2x enhanced lysis buffer and processed through annealing with detector oligos digestion and ligation. Proper amplification was verified using real time PCR green fluorescence. Library purification was performed using the Macherey Nagel NucleoSpin Gel and PCR Cleanup Kit following adjustments detailed in the manufacturer’s instructions. PCR amplification products were pooled into a s…		tissue:whole animal\|genotype:5D WT\|treatment:2 6 diethylnaphthalene\|batch:1	GSM8969885	GSM8969885: 2 6 diethylnaphthalene E; Danio rerio; OTHER	GSM8969885 r1	GSM8969885	1	1mL of 1µm beads and 500µL of lysis buffer were loaded into a round bottom 2mL 96 well plate. 100µL of lysis buffer was added to tube containing pooled embryo samples and 200µL of RNA shield the full contents of the tube were then transferred to the homogenization plate. The plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 275 µL of the supernatant was then transferred to two plates containing 275µL of ethanol. RNA extraction was performed using the KingFisher Apex system. Briefly this process uses Mag beads to collect RNA from the sample post series of washes the RNA is eluted in 50µLof ultra pure water. RNA quality was assessed using the Agilent 4150 TapeStation System. All samples used had RIN scores >9.8. RNA concentration was determined using ThermoFisher Quant iT RNA Reagent and assay kit and ThermoFisher Varioskan ALF multimode plate reader in triplicate samples with high variability between reads were repeated in triplicate. The average of the three quantification reads was used for RNA concentration normalization. Each treatment had 4 5 viable RNA extraction replicates for treatments with 5 viable samples the 4 with the highest concentration and RIN score were used 3 control samples were used from each day group. All 94 samples were normalized to the lowest concentration sample 14.9ng/µL. 1 µL per sample of normalized RNA was used as input for library preparation following manufactures protocol for the 96 sample TempO Seq S1500 + Zebrafish Surrogate Assay panel BioSpyder Technologies Inc. Carlsbad CA United States. Briefly samples were added to the assay plate with an equal volume of 2x enhanced lysis buffer and processed through annealing with detector oligos digestion and ligation. Proper amplification was verified using real time PCR green fluorescence. Library purification was performed using the Macherey Nagel NucleoSpin Gel and PCR Cleanup Kit following adjustments detailed in the manufacturer's instructions. PCR amplification products were pooled into a s…	OTHER	TRANSCRIPTOMIC	other	SINGLE	ILLUMINA	Illumina NovaSeq X	SRP583274			1_E.fastq.gz	fastq	554540646.0	10873346.0	GSM8969885 r1	0:51	A:113384528;C:146196077;G:126957362;T:167877836;N:124843	51				113384528	146196077	126957362	167877836	124843	SRX28685510	SRS24952414	SRA2124446	SARL, Environmental and Molecular Toxicology, Oregon State University	SARL, Environmental and Molecular Toxicology, Oregon State University												B		usable mapping rate	illumina	novaseq_era	unknown	other	unknown	bulk	unknown	unknown	United States	2025-05-06	Multi-stage	Embryo	Whole Organism	All anatomical structures
36073	36073	SRR33444785	SRX28685509	SRS24952412	SRP583274	PRJNA1259243	Structure Dependent Developmental Toxicity of Alkyl Substituted Naphthalenes	GSE296410	Transcriptome Analysis	Naphthalene and its alkyl substituted derivatives are among the most abundant polycyclic aromatic hydrocarbons PAHs in environmental and human exposure studies yet their developmental toxicity and mode of action remain poorly understood due to challenges in testing of semi volatile compounds. This study developed a vial based high through put method to effectively assess the activity of naphthalenes and a set of 24 alkyl substituted naphthalenes. Early life stage zebrafish were exposed to a concentration series of each chemical 0 50 µM in rotating sealed glass vials to minimize volatilization. Benchmark concentration BMC50 values were calculated for morphological endpoints and lowest effect levels were determined for behavioral effects. The data were assessed for evidence of a narcotic mode of action using body burden measurements for select chemicals and logKow modeling. Targeted transcriptomics at a single concentration and timepoint as well as in silico molecular docking were conducted to generate mode of action hypothesis. The vial method enabled detection of highly variable developmental toxicity not previously observed using standard 96 well plate exposures. LogKow and body burden were poor predictors of toxicity suggesting a non narcotic mode of action. Transcriptomic analysis revealed a limited but notable set of differentially expressed genes with evidence for the disruption of glucocorticoid signaling pathways. Molecular docking identified potential protein targets e.g. CYP1A2 NT5E FOLR1 that may mediate observed effects. This study demonstrates the importance of appropriate exposure methods for semi volatile compounds reveals structure dependent toxicity among alkyl substituted naphthalenes and provides a foundation for further mechanistic studies and improved risk assessment of alkyl substituted PAHs. Overall design: Zebrafish were exposed to 25 chemicals naphthalene and 24 alkyl sustituted naphthalenes using a vial exposure method. Embryos were exposed to 20 uM of each chemical in groups of…				2 6 diethylnaphthalene 1D	GSM8969884		source name:whole animal\|tissue:whole animal\|genotype:5D WT\|treatment:2 6 diethylnaphthalene\|batch:1\|geo loc name:missing\|collection date:missing	2 6 diethylnaphthalene 1D	Demultiplexing and alignment was performed using the manufacture provided processing platform TempO SeqR BioSpyder Technologies Inc. Carlsbad CA United States. Counts of genes with multiple probes were summed as instructed by the manufacturer resulting in 3112 unique genes Read counts were normalized by upper quartile normalization using the calcNormFactors function from the Bioconductor package in EdgeR using the 75th percentile of reads per samples as the scaling factor. Principal component analysis PCA of normalized read counts was used to determine that a batch effect was present based on exposure and RNA extraction day nested by experimental design. At this time one control sample control day1 B was identified as an outlier and removed from all future analysis. The batch effect was removed from unnormalized data using the ComBat seq function from the SVA R package Normalzied read counts were normalized by upper quartile normalization using the calcNormFactors function from the Bioconductor package in EdgeR using the 75th percentile of reads per samples as the scaling factor. Assembly: GRCz11 Supplementary files format and content: .csv file containing raw count data Supplementary files format and content: .csv file containing batch corrected and normalized data	whole animal	When the embryos were 6hpf they were bleached in groups of 1000 or less. Embryos were placed in a strainer for transfer between solutions in 500 mL containers. First embryos were placed in system fish water for 1 minute then transferred to a 0.5% sodium chloride solution for 5 minutes then transferred to a % sodium hypochlorite solution for 1 minute and rinsed in EM for 5 minutes and transferred to a clean glass petri dish. Embryos were then loaded into 2mL glass exposure vials in groups of 8 using a glass pasture pipette. Excess EM was removed and 2mL of fresh EM was immediately added. Chemical stocks were diluted to 200 times the desired exposure concentration of 20 µM using DMSO. 10 µL of diluted chemical stock was added to each vial vials were immediately capped and inverted 3 times to ensure mixing no precipitate was observed for any chemicals at the concentrations tested. Vials were loaded into a custom 3D printed vial holder for slow end over end rotation in a dark 28°C incubator overnight. At 24 hours embryos were removed from exposure vials and plated into 96 well plates loaded with 100 µL clean EM. At 48 hpf embryos were collected for RNA extraction embryos from two exposure vials were kept as holdback for morphological assessment. From the remaining wells 6 phenotypically normal embryos were collected and pooled in their vial groups into 1.5 mL Eppendorf tubes placed on ice 30 sec excess media removed placed back on ice 30 sec and 200 µL RNA shield added. Five pooled samples were collected from each treatment.	1mL of 1µm beads and 500µL of lysis buffer were loaded into a round bottom 2mL 96 well plate. 100µL of lysis buffer was added to tube containing pooled embryo samples and 200µL of RNA shield the full contents of the tube were then transferred to the homogenization plate. The plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 275 µL of the supernatant was then transferred to two plates containing 275µL of ethanol. RNA extraction was performed using the KingFisher Apex system. Briefly this process uses Mag beads to collect RNA from the sample post series of washes the RNA is eluted in 50µLof ultra pure water. RNA quality was assessed using the Agilent 4150 TapeStation System. All samples used had RIN scores >9.8. RNA concentration was determined using ThermoFisher Quant iT RNA Reagent and assay kit and ThermoFisher Varioskan ALF multimode plate reader in triplicate samples with high variability between reads were repeated in triplicate. The average of the three quantification reads was used for RNA concentration normalization. Each treatment had 4 5 viable RNA extraction replicates for treatments with 5 viable samples the 4 with the highest concentration and RIN score were used 3 control samples were used from each day group. All 94 samples were normalized to the lowest concentration sample 14.9ng/µL. 1 µL per sample of normalized RNA was used as input for library preparation following manufactures protocol for the 96 sample TempO Seq S1500 + Zebrafish Surrogate Assay panel BioSpyder Technologies Inc. Carlsbad CA United States. Briefly samples were added to the assay plate with an equal volume of 2x enhanced lysis buffer and processed through annealing with detector oligos digestion and ligation. Proper amplification was verified using real time PCR green fluorescence. Library purification was performed using the Macherey Nagel NucleoSpin Gel and PCR Cleanup Kit following adjustments detailed in the manufacturer’s instructions. PCR amplification products were pooled into a s…		tissue:whole animal\|genotype:5D WT\|treatment:2 6 diethylnaphthalene\|batch:1	GSM8969884	GSM8969884: 2 6 diethylnaphthalene 1D; Danio rerio; OTHER	GSM8969884 r1	GSM8969884	1	1mL of 1µm beads and 500µL of lysis buffer were loaded into a round bottom 2mL 96 well plate. 100µL of lysis buffer was added to tube containing pooled embryo samples and 200µL of RNA shield the full contents of the tube were then transferred to the homogenization plate. The plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 275 µL of the supernatant was then transferred to two plates containing 275µL of ethanol. RNA extraction was performed using the KingFisher Apex system. Briefly this process uses Mag beads to collect RNA from the sample post series of washes the RNA is eluted in 50µLof ultra pure water. RNA quality was assessed using the Agilent 4150 TapeStation System. All samples used had RIN scores >9.8. RNA concentration was determined using ThermoFisher Quant iT RNA Reagent and assay kit and ThermoFisher Varioskan ALF multimode plate reader in triplicate samples with high variability between reads were repeated in triplicate. The average of the three quantification reads was used for RNA concentration normalization. Each treatment had 4 5 viable RNA extraction replicates for treatments with 5 viable samples the 4 with the highest concentration and RIN score were used 3 control samples were used from each day group. All 94 samples were normalized to the lowest concentration sample 14.9ng/µL. 1 µL per sample of normalized RNA was used as input for library preparation following manufactures protocol for the 96 sample TempO Seq S1500 + Zebrafish Surrogate Assay panel BioSpyder Technologies Inc. Carlsbad CA United States. Briefly samples were added to the assay plate with an equal volume of 2x enhanced lysis buffer and processed through annealing with detector oligos digestion and ligation. Proper amplification was verified using real time PCR green fluorescence. Library purification was performed using the Macherey Nagel NucleoSpin Gel and PCR Cleanup Kit following adjustments detailed in the manufacturer's instructions. PCR amplification products were pooled into a s…	OTHER	TRANSCRIPTOMIC	other	SINGLE	ILLUMINA	Illumina NovaSeq X	SRP583274			1_D.fastq.gz	fastq	663557685.0	13010935.0	GSM8969884 r1	0:51	A:137450973;C:174284217;G:151682321;T:199990487;N:149687	51				137450973	174284217	151682321	199990487	149687	SRX28685509	SRS24952412	SRA2124446	SARL, Environmental and Molecular Toxicology, Oregon State University	SARL, Environmental and Molecular Toxicology, Oregon State University												B		usable mapping rate	illumina	novaseq_era	unknown	other	unknown	bulk	unknown	unknown	United States	2025-05-06	Multi-stage	Embryo	Whole Organism	All anatomical structures
36074	36074	SRR33444786	SRX28685508	SRS24952410	SRP583274	PRJNA1259243	Structure Dependent Developmental Toxicity of Alkyl Substituted Naphthalenes	GSE296410	Transcriptome Analysis	Naphthalene and its alkyl substituted derivatives are among the most abundant polycyclic aromatic hydrocarbons PAHs in environmental and human exposure studies yet their developmental toxicity and mode of action remain poorly understood due to challenges in testing of semi volatile compounds. This study developed a vial based high through put method to effectively assess the activity of naphthalenes and a set of 24 alkyl substituted naphthalenes. Early life stage zebrafish were exposed to a concentration series of each chemical 0 50 µM in rotating sealed glass vials to minimize volatilization. Benchmark concentration BMC50 values were calculated for morphological endpoints and lowest effect levels were determined for behavioral effects. The data were assessed for evidence of a narcotic mode of action using body burden measurements for select chemicals and logKow modeling. Targeted transcriptomics at a single concentration and timepoint as well as in silico molecular docking were conducted to generate mode of action hypothesis. The vial method enabled detection of highly variable developmental toxicity not previously observed using standard 96 well plate exposures. LogKow and body burden were poor predictors of toxicity suggesting a non narcotic mode of action. Transcriptomic analysis revealed a limited but notable set of differentially expressed genes with evidence for the disruption of glucocorticoid signaling pathways. Molecular docking identified potential protein targets e.g. CYP1A2 NT5E FOLR1 that may mediate observed effects. This study demonstrates the importance of appropriate exposure methods for semi volatile compounds reveals structure dependent toxicity among alkyl substituted naphthalenes and provides a foundation for further mechanistic studies and improved risk assessment of alkyl substituted PAHs. Overall design: Zebrafish were exposed to 25 chemicals naphthalene and 24 alkyl sustituted naphthalenes using a vial exposure method. Embryos were exposed to 20 uM of each chemical in groups of…				2 6 diethylnaphthalene C	GSM8969883		source name:whole animal\|tissue:whole animal\|genotype:5D WT\|treatment:2 6 diethylnaphthalene\|batch:1\|geo loc name:missing\|collection date:missing	2 6 diethylnaphthalene C	Demultiplexing and alignment was performed using the manufacture provided processing platform TempO SeqR BioSpyder Technologies Inc. Carlsbad CA United States. Counts of genes with multiple probes were summed as instructed by the manufacturer resulting in 3112 unique genes Read counts were normalized by upper quartile normalization using the calcNormFactors function from the Bioconductor package in EdgeR using the 75th percentile of reads per samples as the scaling factor. Principal component analysis PCA of normalized read counts was used to determine that a batch effect was present based on exposure and RNA extraction day nested by experimental design. At this time one control sample control day1 B was identified as an outlier and removed from all future analysis. The batch effect was removed from unnormalized data using the ComBat seq function from the SVA R package Normalzied read counts were normalized by upper quartile normalization using the calcNormFactors function from the Bioconductor package in EdgeR using the 75th percentile of reads per samples as the scaling factor. Assembly: GRCz11 Supplementary files format and content: .csv file containing raw count data Supplementary files format and content: .csv file containing batch corrected and normalized data	whole animal	When the embryos were 6hpf they were bleached in groups of 1000 or less. Embryos were placed in a strainer for transfer between solutions in 500 mL containers. First embryos were placed in system fish water for 1 minute then transferred to a 0.5% sodium chloride solution for 5 minutes then transferred to a % sodium hypochlorite solution for 1 minute and rinsed in EM for 5 minutes and transferred to a clean glass petri dish. Embryos were then loaded into 2mL glass exposure vials in groups of 8 using a glass pasture pipette. Excess EM was removed and 2mL of fresh EM was immediately added. Chemical stocks were diluted to 200 times the desired exposure concentration of 20 µM using DMSO. 10 µL of diluted chemical stock was added to each vial vials were immediately capped and inverted 3 times to ensure mixing no precipitate was observed for any chemicals at the concentrations tested. Vials were loaded into a custom 3D printed vial holder for slow end over end rotation in a dark 28°C incubator overnight. At 24 hours embryos were removed from exposure vials and plated into 96 well plates loaded with 100 µL clean EM. At 48 hpf embryos were collected for RNA extraction embryos from two exposure vials were kept as holdback for morphological assessment. From the remaining wells 6 phenotypically normal embryos were collected and pooled in their vial groups into 1.5 mL Eppendorf tubes placed on ice 30 sec excess media removed placed back on ice 30 sec and 200 µL RNA shield added. Five pooled samples were collected from each treatment.	1mL of 1µm beads and 500µL of lysis buffer were loaded into a round bottom 2mL 96 well plate. 100µL of lysis buffer was added to tube containing pooled embryo samples and 200µL of RNA shield the full contents of the tube were then transferred to the homogenization plate. The plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 275 µL of the supernatant was then transferred to two plates containing 275µL of ethanol. RNA extraction was performed using the KingFisher Apex system. Briefly this process uses Mag beads to collect RNA from the sample post series of washes the RNA is eluted in 50µLof ultra pure water. RNA quality was assessed using the Agilent 4150 TapeStation System. All samples used had RIN scores >9.8. RNA concentration was determined using ThermoFisher Quant iT RNA Reagent and assay kit and ThermoFisher Varioskan ALF multimode plate reader in triplicate samples with high variability between reads were repeated in triplicate. The average of the three quantification reads was used for RNA concentration normalization. Each treatment had 4 5 viable RNA extraction replicates for treatments with 5 viable samples the 4 with the highest concentration and RIN score were used 3 control samples were used from each day group. All 94 samples were normalized to the lowest concentration sample 14.9ng/µL. 1 µL per sample of normalized RNA was used as input for library preparation following manufactures protocol for the 96 sample TempO Seq S1500 + Zebrafish Surrogate Assay panel BioSpyder Technologies Inc. Carlsbad CA United States. Briefly samples were added to the assay plate with an equal volume of 2x enhanced lysis buffer and processed through annealing with detector oligos digestion and ligation. Proper amplification was verified using real time PCR green fluorescence. Library purification was performed using the Macherey Nagel NucleoSpin Gel and PCR Cleanup Kit following adjustments detailed in the manufacturer’s instructions. PCR amplification products were pooled into a s…		tissue:whole animal\|genotype:5D WT\|treatment:2 6 diethylnaphthalene\|batch:1	GSM8969883	GSM8969883: 2 6 diethylnaphthalene C; Danio rerio; OTHER	GSM8969883 r1	GSM8969883	1	1mL of 1µm beads and 500µL of lysis buffer were loaded into a round bottom 2mL 96 well plate. 100µL of lysis buffer was added to tube containing pooled embryo samples and 200µL of RNA shield the full contents of the tube were then transferred to the homogenization plate. The plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 275 µL of the supernatant was then transferred to two plates containing 275µL of ethanol. RNA extraction was performed using the KingFisher Apex system. Briefly this process uses Mag beads to collect RNA from the sample post series of washes the RNA is eluted in 50µLof ultra pure water. RNA quality was assessed using the Agilent 4150 TapeStation System. All samples used had RIN scores >9.8. RNA concentration was determined using ThermoFisher Quant iT RNA Reagent and assay kit and ThermoFisher Varioskan ALF multimode plate reader in triplicate samples with high variability between reads were repeated in triplicate. The average of the three quantification reads was used for RNA concentration normalization. Each treatment had 4 5 viable RNA extraction replicates for treatments with 5 viable samples the 4 with the highest concentration and RIN score were used 3 control samples were used from each day group. All 94 samples were normalized to the lowest concentration sample 14.9ng/µL. 1 µL per sample of normalized RNA was used as input for library preparation following manufactures protocol for the 96 sample TempO Seq S1500 + Zebrafish Surrogate Assay panel BioSpyder Technologies Inc. Carlsbad CA United States. Briefly samples were added to the assay plate with an equal volume of 2x enhanced lysis buffer and processed through annealing with detector oligos digestion and ligation. Proper amplification was verified using real time PCR green fluorescence. Library purification was performed using the Macherey Nagel NucleoSpin Gel and PCR Cleanup Kit following adjustments detailed in the manufacturer's instructions. PCR amplification products were pooled into a s…	OTHER	TRANSCRIPTOMIC	other	SINGLE	ILLUMINA	Illumina NovaSeq X	SRP583274			1_C.fastq.gz	fastq	571682562.0	11209462.0	GSM8969883 r1	0:51	A:118075588;C:149868301;G:131235979;T:172374281;N:128413	51				118075588	149868301	131235979	172374281	128413	SRX28685508	SRS24952410	SRA2124446	SARL, Environmental and Molecular Toxicology, Oregon State University	SARL, Environmental and Molecular Toxicology, Oregon State University												B		usable mapping rate	illumina	novaseq_era	unknown	other	unknown	bulk	unknown	unknown	United States	2025-05-06	Multi-stage	Embryo	Whole Organism	All anatomical structures
36075	36075	SRR33444787	SRX28685507	SRS24952411	SRP583274	PRJNA1259243	Structure Dependent Developmental Toxicity of Alkyl Substituted Naphthalenes	GSE296410	Transcriptome Analysis	Naphthalene and its alkyl substituted derivatives are among the most abundant polycyclic aromatic hydrocarbons PAHs in environmental and human exposure studies yet their developmental toxicity and mode of action remain poorly understood due to challenges in testing of semi volatile compounds. This study developed a vial based high through put method to effectively assess the activity of naphthalenes and a set of 24 alkyl substituted naphthalenes. Early life stage zebrafish were exposed to a concentration series of each chemical 0 50 µM in rotating sealed glass vials to minimize volatilization. Benchmark concentration BMC50 values were calculated for morphological endpoints and lowest effect levels were determined for behavioral effects. The data were assessed for evidence of a narcotic mode of action using body burden measurements for select chemicals and logKow modeling. Targeted transcriptomics at a single concentration and timepoint as well as in silico molecular docking were conducted to generate mode of action hypothesis. The vial method enabled detection of highly variable developmental toxicity not previously observed using standard 96 well plate exposures. LogKow and body burden were poor predictors of toxicity suggesting a non narcotic mode of action. Transcriptomic analysis revealed a limited but notable set of differentially expressed genes with evidence for the disruption of glucocorticoid signaling pathways. Molecular docking identified potential protein targets e.g. CYP1A2 NT5E FOLR1 that may mediate observed effects. This study demonstrates the importance of appropriate exposure methods for semi volatile compounds reveals structure dependent toxicity among alkyl substituted naphthalenes and provides a foundation for further mechanistic studies and improved risk assessment of alkyl substituted PAHs. Overall design: Zebrafish were exposed to 25 chemicals naphthalene and 24 alkyl sustituted naphthalenes using a vial exposure method. Embryos were exposed to 20 uM of each chemical in groups of…				2 6 diethylnaphthalene B	GSM8969882		source name:whole animal\|tissue:whole animal\|genotype:5D WT\|treatment:2 6 diethylnaphthalene\|batch:1\|geo loc name:missing\|collection date:missing	2 6 diethylnaphthalene B	Demultiplexing and alignment was performed using the manufacture provided processing platform TempO SeqR BioSpyder Technologies Inc. Carlsbad CA United States. Counts of genes with multiple probes were summed as instructed by the manufacturer resulting in 3112 unique genes Read counts were normalized by upper quartile normalization using the calcNormFactors function from the Bioconductor package in EdgeR using the 75th percentile of reads per samples as the scaling factor. Principal component analysis PCA of normalized read counts was used to determine that a batch effect was present based on exposure and RNA extraction day nested by experimental design. At this time one control sample control day1 B was identified as an outlier and removed from all future analysis. The batch effect was removed from unnormalized data using the ComBat seq function from the SVA R package Normalzied read counts were normalized by upper quartile normalization using the calcNormFactors function from the Bioconductor package in EdgeR using the 75th percentile of reads per samples as the scaling factor. Assembly: GRCz11 Supplementary files format and content: .csv file containing raw count data Supplementary files format and content: .csv file containing batch corrected and normalized data	whole animal	When the embryos were 6hpf they were bleached in groups of 1000 or less. Embryos were placed in a strainer for transfer between solutions in 500 mL containers. First embryos were placed in system fish water for 1 minute then transferred to a 0.5% sodium chloride solution for 5 minutes then transferred to a % sodium hypochlorite solution for 1 minute and rinsed in EM for 5 minutes and transferred to a clean glass petri dish. Embryos were then loaded into 2mL glass exposure vials in groups of 8 using a glass pasture pipette. Excess EM was removed and 2mL of fresh EM was immediately added. Chemical stocks were diluted to 200 times the desired exposure concentration of 20 µM using DMSO. 10 µL of diluted chemical stock was added to each vial vials were immediately capped and inverted 3 times to ensure mixing no precipitate was observed for any chemicals at the concentrations tested. Vials were loaded into a custom 3D printed vial holder for slow end over end rotation in a dark 28°C incubator overnight. At 24 hours embryos were removed from exposure vials and plated into 96 well plates loaded with 100 µL clean EM. At 48 hpf embryos were collected for RNA extraction embryos from two exposure vials were kept as holdback for morphological assessment. From the remaining wells 6 phenotypically normal embryos were collected and pooled in their vial groups into 1.5 mL Eppendorf tubes placed on ice 30 sec excess media removed placed back on ice 30 sec and 200 µL RNA shield added. Five pooled samples were collected from each treatment.	1mL of 1µm beads and 500µL of lysis buffer were loaded into a round bottom 2mL 96 well plate. 100µL of lysis buffer was added to tube containing pooled embryo samples and 200µL of RNA shield the full contents of the tube were then transferred to the homogenization plate. The plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 275 µL of the supernatant was then transferred to two plates containing 275µL of ethanol. RNA extraction was performed using the KingFisher Apex system. Briefly this process uses Mag beads to collect RNA from the sample post series of washes the RNA is eluted in 50µLof ultra pure water. RNA quality was assessed using the Agilent 4150 TapeStation System. All samples used had RIN scores >9.8. RNA concentration was determined using ThermoFisher Quant iT RNA Reagent and assay kit and ThermoFisher Varioskan ALF multimode plate reader in triplicate samples with high variability between reads were repeated in triplicate. The average of the three quantification reads was used for RNA concentration normalization. Each treatment had 4 5 viable RNA extraction replicates for treatments with 5 viable samples the 4 with the highest concentration and RIN score were used 3 control samples were used from each day group. All 94 samples were normalized to the lowest concentration sample 14.9ng/µL. 1 µL per sample of normalized RNA was used as input for library preparation following manufactures protocol for the 96 sample TempO Seq S1500 + Zebrafish Surrogate Assay panel BioSpyder Technologies Inc. Carlsbad CA United States. Briefly samples were added to the assay plate with an equal volume of 2x enhanced lysis buffer and processed through annealing with detector oligos digestion and ligation. Proper amplification was verified using real time PCR green fluorescence. Library purification was performed using the Macherey Nagel NucleoSpin Gel and PCR Cleanup Kit following adjustments detailed in the manufacturer’s instructions. PCR amplification products were pooled into a s…		tissue:whole animal\|genotype:5D WT\|treatment:2 6 diethylnaphthalene\|batch:1	GSM8969882	GSM8969882: 2 6 diethylnaphthalene B; Danio rerio; OTHER	GSM8969882 r1	GSM8969882	1	1mL of 1µm beads and 500µL of lysis buffer were loaded into a round bottom 2mL 96 well plate. 100µL of lysis buffer was added to tube containing pooled embryo samples and 200µL of RNA shield the full contents of the tube were then transferred to the homogenization plate. The plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 275 µL of the supernatant was then transferred to two plates containing 275µL of ethanol. RNA extraction was performed using the KingFisher Apex system. Briefly this process uses Mag beads to collect RNA from the sample post series of washes the RNA is eluted in 50µLof ultra pure water. RNA quality was assessed using the Agilent 4150 TapeStation System. All samples used had RIN scores >9.8. RNA concentration was determined using ThermoFisher Quant iT RNA Reagent and assay kit and ThermoFisher Varioskan ALF multimode plate reader in triplicate samples with high variability between reads were repeated in triplicate. The average of the three quantification reads was used for RNA concentration normalization. Each treatment had 4 5 viable RNA extraction replicates for treatments with 5 viable samples the 4 with the highest concentration and RIN score were used 3 control samples were used from each day group. All 94 samples were normalized to the lowest concentration sample 14.9ng/µL. 1 µL per sample of normalized RNA was used as input for library preparation following manufactures protocol for the 96 sample TempO Seq S1500 + Zebrafish Surrogate Assay panel BioSpyder Technologies Inc. Carlsbad CA United States. Briefly samples were added to the assay plate with an equal volume of 2x enhanced lysis buffer and processed through annealing with detector oligos digestion and ligation. Proper amplification was verified using real time PCR green fluorescence. Library purification was performed using the Macherey Nagel NucleoSpin Gel and PCR Cleanup Kit following adjustments detailed in the manufacturer's instructions. PCR amplification products were pooled into a s…	OTHER	TRANSCRIPTOMIC	other	SINGLE	ILLUMINA	Illumina NovaSeq X	SRP583274			1_B.fastq.gz	fastq	431385387.0	8458537.0	GSM8969882 r1	0:51	A:89066718;C:112472814;G:98920613;T:130827111;N:98131	51				89066718	112472814	98920613	130827111	98131	SRX28685507	SRS24952411	SRA2124446	SARL, Environmental and Molecular Toxicology, Oregon State University	SARL, Environmental and Molecular Toxicology, Oregon State University												B		usable mapping rate	illumina	novaseq_era	unknown	other	unknown	bulk	unknown	unknown	United States	2025-05-06	Multi-stage	Embryo	Whole Organism	All anatomical structures
36076	36076	SRR33444788	SRX28685506	SRS24952409	SRP583274	PRJNA1259243	Structure Dependent Developmental Toxicity of Alkyl Substituted Naphthalenes	GSE296410	Transcriptome Analysis	Naphthalene and its alkyl substituted derivatives are among the most abundant polycyclic aromatic hydrocarbons PAHs in environmental and human exposure studies yet their developmental toxicity and mode of action remain poorly understood due to challenges in testing of semi volatile compounds. This study developed a vial based high through put method to effectively assess the activity of naphthalenes and a set of 24 alkyl substituted naphthalenes. Early life stage zebrafish were exposed to a concentration series of each chemical 0 50 µM in rotating sealed glass vials to minimize volatilization. Benchmark concentration BMC50 values were calculated for morphological endpoints and lowest effect levels were determined for behavioral effects. The data were assessed for evidence of a narcotic mode of action using body burden measurements for select chemicals and logKow modeling. Targeted transcriptomics at a single concentration and timepoint as well as in silico molecular docking were conducted to generate mode of action hypothesis. The vial method enabled detection of highly variable developmental toxicity not previously observed using standard 96 well plate exposures. LogKow and body burden were poor predictors of toxicity suggesting a non narcotic mode of action. Transcriptomic analysis revealed a limited but notable set of differentially expressed genes with evidence for the disruption of glucocorticoid signaling pathways. Molecular docking identified potential protein targets e.g. CYP1A2 NT5E FOLR1 that may mediate observed effects. This study demonstrates the importance of appropriate exposure methods for semi volatile compounds reveals structure dependent toxicity among alkyl substituted naphthalenes and provides a foundation for further mechanistic studies and improved risk assessment of alkyl substituted PAHs. Overall design: Zebrafish were exposed to 25 chemicals naphthalene and 24 alkyl sustituted naphthalenes using a vial exposure method. Embryos were exposed to 20 uM of each chemical in groups of…				2 4 5 trimethylnaphthalene E	GSM8969921		source name:whole animal\|tissue:whole animal\|genotype:5D WT\|treatment:2 4 5 trimethylnaphthalene\|batch:1\|geo loc name:missing\|collection date:missing	2 4 5 trimethylnaphthalene E	Demultiplexing and alignment was performed using the manufacture provided processing platform TempO SeqR BioSpyder Technologies Inc. Carlsbad CA United States. Counts of genes with multiple probes were summed as instructed by the manufacturer resulting in 3112 unique genes Read counts were normalized by upper quartile normalization using the calcNormFactors function from the Bioconductor package in EdgeR using the 75th percentile of reads per samples as the scaling factor. Principal component analysis PCA of normalized read counts was used to determine that a batch effect was present based on exposure and RNA extraction day nested by experimental design. At this time one control sample control day1 B was identified as an outlier and removed from all future analysis. The batch effect was removed from unnormalized data using the ComBat seq function from the SVA R package Normalzied read counts were normalized by upper quartile normalization using the calcNormFactors function from the Bioconductor package in EdgeR using the 75th percentile of reads per samples as the scaling factor. Assembly: GRCz11 Supplementary files format and content: .csv file containing raw count data Supplementary files format and content: .csv file containing batch corrected and normalized data	whole animal	When the embryos were 6hpf they were bleached in groups of 1000 or less. Embryos were placed in a strainer for transfer between solutions in 500 mL containers. First embryos were placed in system fish water for 1 minute then transferred to a 0.5% sodium chloride solution for 5 minutes then transferred to a % sodium hypochlorite solution for 1 minute and rinsed in EM for 5 minutes and transferred to a clean glass petri dish. Embryos were then loaded into 2mL glass exposure vials in groups of 8 using a glass pasture pipette. Excess EM was removed and 2mL of fresh EM was immediately added. Chemical stocks were diluted to 200 times the desired exposure concentration of 20 µM using DMSO. 10 µL of diluted chemical stock was added to each vial vials were immediately capped and inverted 3 times to ensure mixing no precipitate was observed for any chemicals at the concentrations tested. Vials were loaded into a custom 3D printed vial holder for slow end over end rotation in a dark 28°C incubator overnight. At 24 hours embryos were removed from exposure vials and plated into 96 well plates loaded with 100 µL clean EM. At 48 hpf embryos were collected for RNA extraction embryos from two exposure vials were kept as holdback for morphological assessment. From the remaining wells 6 phenotypically normal embryos were collected and pooled in their vial groups into 1.5 mL Eppendorf tubes placed on ice 30 sec excess media removed placed back on ice 30 sec and 200 µL RNA shield added. Five pooled samples were collected from each treatment.	1mL of 1µm beads and 500µL of lysis buffer were loaded into a round bottom 2mL 96 well plate. 100µL of lysis buffer was added to tube containing pooled embryo samples and 200µL of RNA shield the full contents of the tube were then transferred to the homogenization plate. The plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 275 µL of the supernatant was then transferred to two plates containing 275µL of ethanol. RNA extraction was performed using the KingFisher Apex system. Briefly this process uses Mag beads to collect RNA from the sample post series of washes the RNA is eluted in 50µLof ultra pure water. RNA quality was assessed using the Agilent 4150 TapeStation System. All samples used had RIN scores >9.8. RNA concentration was determined using ThermoFisher Quant iT RNA Reagent and assay kit and ThermoFisher Varioskan ALF multimode plate reader in triplicate samples with high variability between reads were repeated in triplicate. The average of the three quantification reads was used for RNA concentration normalization. Each treatment had 4 5 viable RNA extraction replicates for treatments with 5 viable samples the 4 with the highest concentration and RIN score were used 3 control samples were used from each day group. All 94 samples were normalized to the lowest concentration sample 14.9ng/µL. 1 µL per sample of normalized RNA was used as input for library preparation following manufactures protocol for the 96 sample TempO Seq S1500 + Zebrafish Surrogate Assay panel BioSpyder Technologies Inc. Carlsbad CA United States. Briefly samples were added to the assay plate with an equal volume of 2x enhanced lysis buffer and processed through annealing with detector oligos digestion and ligation. Proper amplification was verified using real time PCR green fluorescence. Library purification was performed using the Macherey Nagel NucleoSpin Gel and PCR Cleanup Kit following adjustments detailed in the manufacturer’s instructions. PCR amplification products were pooled into a s…		tissue:whole animal\|genotype:5D WT\|treatment:2 4 5 trimethylnaphthalene\|batch:1	GSM8969921	GSM8969921: 2 4 5 trimethylnaphthalene E; Danio rerio; OTHER	GSM8969921 r1	GSM8969921	1	1mL of 1µm beads and 500µL of lysis buffer were loaded into a round bottom 2mL 96 well plate. 100µL of lysis buffer was added to tube containing pooled embryo samples and 200µL of RNA shield the full contents of the tube were then transferred to the homogenization plate. The plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 275 µL of the supernatant was then transferred to two plates containing 275µL of ethanol. RNA extraction was performed using the KingFisher Apex system. Briefly this process uses Mag beads to collect RNA from the sample post series of washes the RNA is eluted in 50µLof ultra pure water. RNA quality was assessed using the Agilent 4150 TapeStation System. All samples used had RIN scores >9.8. RNA concentration was determined using ThermoFisher Quant iT RNA Reagent and assay kit and ThermoFisher Varioskan ALF multimode plate reader in triplicate samples with high variability between reads were repeated in triplicate. The average of the three quantification reads was used for RNA concentration normalization. Each treatment had 4 5 viable RNA extraction replicates for treatments with 5 viable samples the 4 with the highest concentration and RIN score were used 3 control samples were used from each day group. All 94 samples were normalized to the lowest concentration sample 14.9ng/µL. 1 µL per sample of normalized RNA was used as input for library preparation following manufactures protocol for the 96 sample TempO Seq S1500 + Zebrafish Surrogate Assay panel BioSpyder Technologies Inc. Carlsbad CA United States. Briefly samples were added to the assay plate with an equal volume of 2x enhanced lysis buffer and processed through annealing with detector oligos digestion and ligation. Proper amplification was verified using real time PCR green fluorescence. Library purification was performed using the Macherey Nagel NucleoSpin Gel and PCR Cleanup Kit following adjustments detailed in the manufacturer's instructions. PCR amplification products were pooled into a s…	OTHER	TRANSCRIPTOMIC	other	SINGLE	ILLUMINA	Illumina NovaSeq X	SRP583274			7_E.fastq.gz	fastq	674676552.0	13228952.0	GSM8969921 r1	0:51	A:137983580;C:176549665;G:153689206;T:206301698;N:152403	51				137983580	176549665	153689206	206301698	152403	SRX28685506	SRS24952409	SRA2124446	SARL, Environmental and Molecular Toxicology, Oregon State University	SARL, Environmental and Molecular Toxicology, Oregon State University												B		usable mapping rate	illumina	novaseq_era	unknown	other	unknown	bulk	unknown	unknown	United States	2025-05-06	Multi-stage	Embryo	Whole Organism	All anatomical structures
36077	36077	SRR33444789	SRX28685505	SRS24952408	SRP583274	PRJNA1259243	Structure Dependent Developmental Toxicity of Alkyl Substituted Naphthalenes	GSE296410	Transcriptome Analysis	Naphthalene and its alkyl substituted derivatives are among the most abundant polycyclic aromatic hydrocarbons PAHs in environmental and human exposure studies yet their developmental toxicity and mode of action remain poorly understood due to challenges in testing of semi volatile compounds. This study developed a vial based high through put method to effectively assess the activity of naphthalenes and a set of 24 alkyl substituted naphthalenes. Early life stage zebrafish were exposed to a concentration series of each chemical 0 50 µM in rotating sealed glass vials to minimize volatilization. Benchmark concentration BMC50 values were calculated for morphological endpoints and lowest effect levels were determined for behavioral effects. The data were assessed for evidence of a narcotic mode of action using body burden measurements for select chemicals and logKow modeling. Targeted transcriptomics at a single concentration and timepoint as well as in silico molecular docking were conducted to generate mode of action hypothesis. The vial method enabled detection of highly variable developmental toxicity not previously observed using standard 96 well plate exposures. LogKow and body burden were poor predictors of toxicity suggesting a non narcotic mode of action. Transcriptomic analysis revealed a limited but notable set of differentially expressed genes with evidence for the disruption of glucocorticoid signaling pathways. Molecular docking identified potential protein targets e.g. CYP1A2 NT5E FOLR1 that may mediate observed effects. This study demonstrates the importance of appropriate exposure methods for semi volatile compounds reveals structure dependent toxicity among alkyl substituted naphthalenes and provides a foundation for further mechanistic studies and improved risk assessment of alkyl substituted PAHs. Overall design: Zebrafish were exposed to 25 chemicals naphthalene and 24 alkyl sustituted naphthalenes using a vial exposure method. Embryos were exposed to 20 uM of each chemical in groups of…				2 4 5 trimethylnaphthalene D	GSM8969920		source name:whole animal\|tissue:whole animal\|genotype:5D WT\|treatment:2 4 5 trimethylnaphthalene\|batch:1\|geo loc name:missing\|collection date:missing	2 4 5 trimethylnaphthalene D	Demultiplexing and alignment was performed using the manufacture provided processing platform TempO SeqR BioSpyder Technologies Inc. Carlsbad CA United States. Counts of genes with multiple probes were summed as instructed by the manufacturer resulting in 3112 unique genes Read counts were normalized by upper quartile normalization using the calcNormFactors function from the Bioconductor package in EdgeR using the 75th percentile of reads per samples as the scaling factor. Principal component analysis PCA of normalized read counts was used to determine that a batch effect was present based on exposure and RNA extraction day nested by experimental design. At this time one control sample control day1 B was identified as an outlier and removed from all future analysis. The batch effect was removed from unnormalized data using the ComBat seq function from the SVA R package Normalzied read counts were normalized by upper quartile normalization using the calcNormFactors function from the Bioconductor package in EdgeR using the 75th percentile of reads per samples as the scaling factor. Assembly: GRCz11 Supplementary files format and content: .csv file containing raw count data Supplementary files format and content: .csv file containing batch corrected and normalized data	whole animal	When the embryos were 6hpf they were bleached in groups of 1000 or less. Embryos were placed in a strainer for transfer between solutions in 500 mL containers. First embryos were placed in system fish water for 1 minute then transferred to a 0.5% sodium chloride solution for 5 minutes then transferred to a % sodium hypochlorite solution for 1 minute and rinsed in EM for 5 minutes and transferred to a clean glass petri dish. Embryos were then loaded into 2mL glass exposure vials in groups of 8 using a glass pasture pipette. Excess EM was removed and 2mL of fresh EM was immediately added. Chemical stocks were diluted to 200 times the desired exposure concentration of 20 µM using DMSO. 10 µL of diluted chemical stock was added to each vial vials were immediately capped and inverted 3 times to ensure mixing no precipitate was observed for any chemicals at the concentrations tested. Vials were loaded into a custom 3D printed vial holder for slow end over end rotation in a dark 28°C incubator overnight. At 24 hours embryos were removed from exposure vials and plated into 96 well plates loaded with 100 µL clean EM. At 48 hpf embryos were collected for RNA extraction embryos from two exposure vials were kept as holdback for morphological assessment. From the remaining wells 6 phenotypically normal embryos were collected and pooled in their vial groups into 1.5 mL Eppendorf tubes placed on ice 30 sec excess media removed placed back on ice 30 sec and 200 µL RNA shield added. Five pooled samples were collected from each treatment.	1mL of 1µm beads and 500µL of lysis buffer were loaded into a round bottom 2mL 96 well plate. 100µL of lysis buffer was added to tube containing pooled embryo samples and 200µL of RNA shield the full contents of the tube were then transferred to the homogenization plate. The plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 275 µL of the supernatant was then transferred to two plates containing 275µL of ethanol. RNA extraction was performed using the KingFisher Apex system. Briefly this process uses Mag beads to collect RNA from the sample post series of washes the RNA is eluted in 50µLof ultra pure water. RNA quality was assessed using the Agilent 4150 TapeStation System. All samples used had RIN scores >9.8. RNA concentration was determined using ThermoFisher Quant iT RNA Reagent and assay kit and ThermoFisher Varioskan ALF multimode plate reader in triplicate samples with high variability between reads were repeated in triplicate. The average of the three quantification reads was used for RNA concentration normalization. Each treatment had 4 5 viable RNA extraction replicates for treatments with 5 viable samples the 4 with the highest concentration and RIN score were used 3 control samples were used from each day group. All 94 samples were normalized to the lowest concentration sample 14.9ng/µL. 1 µL per sample of normalized RNA was used as input for library preparation following manufactures protocol for the 96 sample TempO Seq S1500 + Zebrafish Surrogate Assay panel BioSpyder Technologies Inc. Carlsbad CA United States. Briefly samples were added to the assay plate with an equal volume of 2x enhanced lysis buffer and processed through annealing with detector oligos digestion and ligation. Proper amplification was verified using real time PCR green fluorescence. Library purification was performed using the Macherey Nagel NucleoSpin Gel and PCR Cleanup Kit following adjustments detailed in the manufacturer’s instructions. PCR amplification products were pooled into a s…		tissue:whole animal\|genotype:5D WT\|treatment:2 4 5 trimethylnaphthalene\|batch:1	GSM8969920	GSM8969920: 2 4 5 trimethylnaphthalene D; Danio rerio; OTHER	GSM8969920 r1	GSM8969920	1	1mL of 1µm beads and 500µL of lysis buffer were loaded into a round bottom 2mL 96 well plate. 100µL of lysis buffer was added to tube containing pooled embryo samples and 200µL of RNA shield the full contents of the tube were then transferred to the homogenization plate. The plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 275 µL of the supernatant was then transferred to two plates containing 275µL of ethanol. RNA extraction was performed using the KingFisher Apex system. Briefly this process uses Mag beads to collect RNA from the sample post series of washes the RNA is eluted in 50µLof ultra pure water. RNA quality was assessed using the Agilent 4150 TapeStation System. All samples used had RIN scores >9.8. RNA concentration was determined using ThermoFisher Quant iT RNA Reagent and assay kit and ThermoFisher Varioskan ALF multimode plate reader in triplicate samples with high variability between reads were repeated in triplicate. The average of the three quantification reads was used for RNA concentration normalization. Each treatment had 4 5 viable RNA extraction replicates for treatments with 5 viable samples the 4 with the highest concentration and RIN score were used 3 control samples were used from each day group. All 94 samples were normalized to the lowest concentration sample 14.9ng/µL. 1 µL per sample of normalized RNA was used as input for library preparation following manufactures protocol for the 96 sample TempO Seq S1500 + Zebrafish Surrogate Assay panel BioSpyder Technologies Inc. Carlsbad CA United States. Briefly samples were added to the assay plate with an equal volume of 2x enhanced lysis buffer and processed through annealing with detector oligos digestion and ligation. Proper amplification was verified using real time PCR green fluorescence. Library purification was performed using the Macherey Nagel NucleoSpin Gel and PCR Cleanup Kit following adjustments detailed in the manufacturer's instructions. PCR amplification products were pooled into a s…	OTHER	TRANSCRIPTOMIC	other	SINGLE	ILLUMINA	Illumina NovaSeq X	SRP583274			7_D.fastq.gz	fastq	585836439.0	11486989.0	GSM8969920 r1	0:51	A:119799324;C:153323440;G:134779037;T:177801818;N:132820	51				119799324	153323440	134779037	177801818	132820	SRX28685505	SRS24952408	SRA2124446	SARL, Environmental and Molecular Toxicology, Oregon State University	SARL, Environmental and Molecular Toxicology, Oregon State University												B		usable mapping rate	illumina	novaseq_era	unknown	other	unknown	bulk	unknown	unknown	United States	2025-05-06	Multi-stage	Embryo	Whole Organism	All anatomical structures

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CREATE TABLE run_metadata("run.accession" VARCHAR, "experiment.accession" VARCHAR, "sample.accession" VARCHAR, "study.accession" VARCHAR, bioproject VARCHAR, "study.title" VARCHAR, "study.alias" VARCHAR, "study.type" VARCHAR, "study.abstract" VARCHAR, "study.attributes" VARCHAR, "study.PMIDs" VARCHAR, "sample.description" VARCHAR, "sample.title" VARCHAR, "sample.alias" VARCHAR, "sample.centername" VARCHAR, "sample.attributes" VARCHAR, "GEOsample.title" VARCHAR, "GEOsample.dataprocessing" VARCHAR, "GEOsample.source" VARCHAR, "GEOsample.treatmentprotocol" VARCHAR, "GEOsample.extractprotocol" VARCHAR, "GEOsample.growthprotocol" VARCHAR, "GEOsample.characteristics" VARCHAR, "GEOsample.accession" VARCHAR, "experiment.title" VARCHAR, "experiment.alias" VARCHAR, "experiment.library_name" VARCHAR, "experiment.design_description" VARCHAR, "experiment.library_construction_protocol" VARCHAR, "experiment.attributes" VARCHAR, "experiment.library_strategy" VARCHAR, "experiment.library_source" VARCHAR, "experiment.library_selection" VARCHAR, "experiment.library_layout" VARCHAR, "experiment.platform" VARCHAR, "experiment.instrument_model" VARCHAR, "experiment.spot_descriptor" VARCHAR, "experiment.study_ref" VARCHAR, "run.title" VARCHAR, "run.attributes" VARCHAR, "run.filename" VARCHAR, "run.semantic_name" VARCHAR, "run.total_bases" DOUBLE, "run.total_spots" DOUBLE, "run.alias" VARCHAR, "run.read_lengths" VARCHAR, "run.base_counts" VARCHAR, "run.r1_length" BIGINT, "run.r2_length" BIGINT, "run.r3_length" BIGINT, "run.r4_length" BIGINT, "run.Acount" BIGINT, "run.Ccount" BIGINT, "run.Gcount" BIGINT, "run.Tcount" BIGINT, "run.Ncount" BIGINT, "run.experiment" VARCHAR, "run.pool_member" VARCHAR, "submission.accession" VARCHAR, "submission.srasource" VARCHAR, "submission.bioprojectsource" VARCHAR, "seqdetective.n_mates" BIGINT, "seqdetective.mapping_rate.mate1" DOUBLE, "seqdetective.mapping_rate.mate2" DOUBLE, "seqdetective.nofeature_rate.mate1" DOUBLE, "seqdetective.nofeature_rate.mate2" DOUBLE, "seqdetective.sparsity.mate1" DOUBLE, "seqdetective.sparsity.mate2" DOUBLE, "seqdetective.pos_strand_rate.mate1" DOUBLE, "seqdetective.pos_strand_rate.mate2" DOUBLE, "seqdetective.readlen.mate1" BIGINT, "seqdetective.readlen.mate2" BIGINT, "seqdetective.judgement.mate1" VARCHAR, "seqdetective.judgement.mate2" VARCHAR, "seqdetective.judgement.reason" VARCHAR, platform_family VARCHAR, instrument_generation VARCHAR, read_bias VARCHAR, selection_class VARCHAR, prep_kit VARCHAR, sc_or_bulk VARCHAR, tech_class VARCHAR, technology VARCHAR, tech_variant VARCHAR, "submission.bioprojectsource.country" VARCHAR, earliest_date DATE, devstage_curation VARCHAR, devstage_curation_coarse VARCHAR, tissue_curation VARCHAR, tissue_curation_coarse VARCHAR);;
CREATE INDEX idx_run_bioproject ON run_metadata(bioproject);;
CREATE INDEX idx_run_run_accession ON run_metadata("run.accession");;