rowid,run.accession,experiment.accession,sample.accession,study.accession,bioproject,study.title,study.alias,study.type,study.abstract,study.attributes,study.PMIDs,sample.description,sample.title,sample.alias,sample.centername,sample.attributes,GEOsample.title,GEOsample.dataprocessing,GEOsample.source,GEOsample.treatmentprotocol,GEOsample.extractprotocol,GEOsample.growthprotocol,GEOsample.characteristics,GEOsample.accession,experiment.title,experiment.alias,experiment.library_name,experiment.design_description,experiment.library_construction_protocol,experiment.attributes,experiment.library_strategy,experiment.library_source,experiment.library_selection,experiment.library_layout,experiment.platform,experiment.instrument_model,experiment.spot_descriptor,experiment.study_ref,run.title,run.attributes,run.filename,run.semantic_name,run.total_bases,run.total_spots,run.alias,run.read_lengths,run.base_counts,run.r1_length,run.r2_length,run.r3_length,run.r4_length,run.Acount,run.Ccount,run.Gcount,run.Tcount,run.Ncount,run.experiment,run.pool_member,submission.accession,submission.srasource,submission.bioprojectsource,seqdetective.n_mates,seqdetective.mapping_rate.mate1,seqdetective.mapping_rate.mate2,seqdetective.nofeature_rate.mate1,seqdetective.nofeature_rate.mate2,seqdetective.sparsity.mate1,seqdetective.sparsity.mate2,seqdetective.pos_strand_rate.mate1,seqdetective.pos_strand_rate.mate2,seqdetective.readlen.mate1,seqdetective.readlen.mate2,seqdetective.judgement.mate1,seqdetective.judgement.mate2,seqdetective.judgement.reason,platform_family,instrument_generation,read_bias,selection_class,prep_kit,sc_or_bulk,tech_class,technology,tech_variant,submission.bioprojectsource.country,earliest_date,devstage_curation,devstage_curation_coarse,tissue_curation,tissue_curation_coarse
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,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,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,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 supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 µl 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 µl 2x SSC. The RNA was eluted from the beads first with 100 µl 10 mM HEPES pH 7.5 at 65°C for 3 min and then second with 100 µl water at 65°C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 µl water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/µl T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23°C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 µl water. The ligated RNA was mixed with 0.6 µl 100 µM reverse transcription primer KXS037 in a total volume of 12 µl  incubated at 65°C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 µl reaction containing 1x First Strand Buffer  500 µM dNTPs  5 mM dithiothreitol  1 unit/µl SUPERase•In  and 200 units SuperScript III Thermo Fisher  18080044 at 50°C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min  followed by neutralization with 36.7 µl 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer’s suggested protocol for 10–15 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3ʹ adapter KXS013 was used for 3ʹ end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 µl. The cDNAs and the oligo were annealed by incubation at 65°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 100 µl MyOne Streptavidin C1 beads and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 µl 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 µl water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.,,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 supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 µl 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 µl 2x SSC. The RNA was eluted from the beads first with 100 µl 10 mM HEPES pH 7.5 at 65°C for 3 min and then second with 100 µl water at 65°C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 µl water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/µl T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23°C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 µl water. The ligated RNA was mixed with 0.6 µl 100 µM reverse transcription primer KXS037 in a total volume of 12 µl  incubated at 65°C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 µl reaction containing 1x First Strand Buffer  500 µM dNTPs  5 mM dithiothreitol  1 unit/µl SUPERase•In  and 200 units SuperScript III Thermo Fisher  18080044 at 50°C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min  followed by neutralization with 36.7 µl 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer's suggested protocol for 10–15 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3ʹ adapter KXS013 was used for 3ʹ end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 µl. The cDNAs and the oligo were annealed by incubation at 65°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 100 µl MyOne Streptavidin C1 beads and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 µl 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 µl water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.,,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,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 supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 µl 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 µl 2x SSC. The RNA was eluted from the beads first with 100 µl 10 mM HEPES pH 7.5 at 65°C for 3 min and then second with 100 µl water at 65°C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 µl water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/µl T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23°C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 µl water. The ligated RNA was mixed with 0.6 µl 100 µM reverse transcription primer KXS037 in a total volume of 12 µl  incubated at 65°C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 µl reaction containing 1x First Strand Buffer  500 µM dNTPs  5 mM dithiothreitol  1 unit/µl SUPERase•In  and 200 units SuperScript III Thermo Fisher  18080044 at 50°C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min  followed by neutralization with 36.7 µl 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer’s suggested protocol for 10–15 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3ʹ adapter KXS013 was used for 3ʹ end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 µl. The cDNAs and the oligo were annealed by incubation at 65°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 100 µl MyOne Streptavidin C1 beads and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 µl 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 µl water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.,,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 supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 µl 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 µl 2x SSC. The RNA was eluted from the beads first with 100 µl 10 mM HEPES pH 7.5 at 65°C for 3 min and then second with 100 µl water at 65°C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 µl water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/µl T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23°C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 µl water. The ligated RNA was mixed with 0.6 µl 100 µM reverse transcription primer KXS037 in a total volume of 12 µl  incubated at 65°C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 µl reaction containing 1x First Strand Buffer  500 µM dNTPs  5 mM dithiothreitol  1 unit/µl SUPERase•In  and 200 units SuperScript III Thermo Fisher  18080044 at 50°C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min  followed by neutralization with 36.7 µl 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer's suggested protocol for 10–15 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3ʹ adapter KXS013 was used for 3ʹ end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 µl. The cDNAs and the oligo were annealed by incubation at 65°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 100 µl MyOne Streptavidin C1 beads and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 µl 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 µl water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.,,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,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 supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 µl 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 µl 2x SSC. The RNA was eluted from the beads first with 100 µl 10 mM HEPES pH 7.5 at 65°C for 3 min and then second with 100 µl water at 65°C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 µl water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/µl T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23°C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 µl water. The ligated RNA was mixed with 0.6 µl 100 µM reverse transcription primer KXS037 in a total volume of 12 µl  incubated at 65°C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 µl reaction containing 1x First Strand Buffer  500 µM dNTPs  5 mM dithiothreitol  1 unit/µl SUPERase•In  and 200 units SuperScript III Thermo Fisher  18080044 at 50°C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min  followed by neutralization with 36.7 µl 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer’s suggested protocol for 10–15 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3ʹ adapter KXS013 was used for 3ʹ end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 µl. The cDNAs and the oligo were annealed by incubation at 65°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 100 µl MyOne Streptavidin C1 beads and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 µl 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 µl water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.,,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 supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 µl 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 µl 2x SSC. The RNA was eluted from the beads first with 100 µl 10 mM HEPES pH 7.5 at 65°C for 3 min and then second with 100 µl water at 65°C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 µl water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/µl T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23°C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 µl water. The ligated RNA was mixed with 0.6 µl 100 µM reverse transcription primer KXS037 in a total volume of 12 µl  incubated at 65°C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 µl reaction containing 1x First Strand Buffer  500 µM dNTPs  5 mM dithiothreitol  1 unit/µl SUPERase•In  and 200 units SuperScript III Thermo Fisher  18080044 at 50°C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min  followed by neutralization with 36.7 µl 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer's suggested protocol for 10–15 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3ʹ adapter KXS013 was used for 3ʹ end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 µl. The cDNAs and the oligo were annealed by incubation at 65°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 100 µl MyOne Streptavidin C1 beads and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 µl 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 µl water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.,,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,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 supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 µl 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 µl 2x SSC. The RNA was eluted from the beads first with 100 µl 10 mM HEPES pH 7.5 at 65°C for 3 min and then second with 100 µl water at 65°C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 µl water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/µl T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23°C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 µl water. The ligated RNA was mixed with 0.6 µl 100 µM reverse transcription primer KXS037 in a total volume of 12 µl  incubated at 65°C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 µl reaction containing 1x First Strand Buffer  500 µM dNTPs  5 mM dithiothreitol  1 unit/µl SUPERase•In  and 200 units SuperScript III Thermo Fisher  18080044 at 50°C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min  followed by neutralization with 36.7 µl 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer’s suggested protocol for 10–15 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3ʹ adapter KXS013 was used for 3ʹ end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 µl. The cDNAs and the oligo were annealed by incubation at 65°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 100 µl MyOne Streptavidin C1 beads and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 µl 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 µl water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.,,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 supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 µl 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 µl 2x SSC. The RNA was eluted from the beads first with 100 µl 10 mM HEPES pH 7.5 at 65°C for 3 min and then second with 100 µl water at 65°C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 µl water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/µl T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23°C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 µl water. The ligated RNA was mixed with 0.6 µl 100 µM reverse transcription primer KXS037 in a total volume of 12 µl  incubated at 65°C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 µl reaction containing 1x First Strand Buffer  500 µM dNTPs  5 mM dithiothreitol  1 unit/µl SUPERase•In  and 200 units SuperScript III Thermo Fisher  18080044 at 50°C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min  followed by neutralization with 36.7 µl 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer's suggested protocol for 10–15 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3ʹ adapter KXS013 was used for 3ʹ end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 µl. The cDNAs and the oligo were annealed by incubation at 65°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 100 µl MyOne Streptavidin C1 beads and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 µl 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 µl water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.,,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,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 supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 µl 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 µl 2x SSC. The RNA was eluted from the beads first with 100 µl 10 mM HEPES pH 7.5 at 65°C for 3 min and then second with 100 µl water at 65°C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 µl water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/µl T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23°C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 µl water. The ligated RNA was mixed with 0.6 µl 100 µM reverse transcription primer KXS037 in a total volume of 12 µl  incubated at 65°C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 µl reaction containing 1x First Strand Buffer  500 µM dNTPs  5 mM dithiothreitol  1 unit/µl SUPERase•In  and 200 units SuperScript III Thermo Fisher  18080044 at 50°C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min  followed by neutralization with 36.7 µl 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer’s suggested protocol for 10–15 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3ʹ adapter KXS013 was used for 3ʹ end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 µl. The cDNAs and the oligo were annealed by incubation at 65°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 100 µl MyOne Streptavidin C1 beads and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 µl 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 µl water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.,,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 supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 µl 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 µl 2x SSC. The RNA was eluted from the beads first with 100 µl 10 mM HEPES pH 7.5 at 65°C for 3 min and then second with 100 µl water at 65°C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 µl water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/µl T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23°C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 µl water. The ligated RNA was mixed with 0.6 µl 100 µM reverse transcription primer KXS037 in a total volume of 12 µl  incubated at 65°C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 µl reaction containing 1x First Strand Buffer  500 µM dNTPs  5 mM dithiothreitol  1 unit/µl SUPERase•In  and 200 units SuperScript III Thermo Fisher  18080044 at 50°C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min  followed by neutralization with 36.7 µl 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer's suggested protocol for 10–15 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3ʹ adapter KXS013 was used for 3ʹ end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 µl. The cDNAs and the oligo were annealed by incubation at 65°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 100 µl MyOne Streptavidin C1 beads and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 µl 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 µl water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.,,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,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 supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 µl 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 µl 2x SSC. The RNA was eluted from the beads first with 100 µl 10 mM HEPES pH 7.5 at 65°C for 3 min and then second with 100 µl water at 65°C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 µl water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/µl T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23°C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 µl water. The ligated RNA was mixed with 0.6 µl 100 µM reverse transcription primer KXS037 in a total volume of 12 µl  incubated at 65°C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 µl reaction containing 1x First Strand Buffer  500 µM dNTPs  5 mM dithiothreitol  1 unit/µl SUPERase•In  and 200 units SuperScript III Thermo Fisher  18080044 at 50°C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min  followed by neutralization with 36.7 µl 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer’s suggested protocol for 10–15 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3ʹ adapter KXS013 was used for 3ʹ end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 µl. The cDNAs and the oligo were annealed by incubation at 65°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 100 µl MyOne Streptavidin C1 beads and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 µl 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 µl water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.,,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 supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 µl 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 µl 2x SSC. The RNA was eluted from the beads first with 100 µl 10 mM HEPES pH 7.5 at 65°C for 3 min and then second with 100 µl water at 65°C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 µl water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/µl T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23°C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 µl water. The ligated RNA was mixed with 0.6 µl 100 µM reverse transcription primer KXS037 in a total volume of 12 µl  incubated at 65°C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 µl reaction containing 1x First Strand Buffer  500 µM dNTPs  5 mM dithiothreitol  1 unit/µl SUPERase•In  and 200 units SuperScript III Thermo Fisher  18080044 at 50°C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min  followed by neutralization with 36.7 µl 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer's suggested protocol for 10–15 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3ʹ adapter KXS013 was used for 3ʹ end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 µl. The cDNAs and the oligo were annealed by incubation at 65°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 100 µl MyOne Streptavidin C1 beads and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 µl 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 µl water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.,,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,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 supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 µl 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 µl 2x SSC. The RNA was eluted from the beads first with 100 µl 10 mM HEPES pH 7.5 at 65°C for 3 min and then second with 100 µl water at 65°C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 µl water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/µl T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23°C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 µl water. The ligated RNA was mixed with 0.6 µl 100 µM reverse transcription primer KXS037 in a total volume of 12 µl  incubated at 65°C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 µl reaction containing 1x First Strand Buffer  500 µM dNTPs  5 mM dithiothreitol  1 unit/µl SUPERase•In  and 200 units SuperScript III Thermo Fisher  18080044 at 50°C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min  followed by neutralization with 36.7 µl 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer’s suggested protocol for 10–15 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3ʹ adapter KXS013 was used for 3ʹ end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 µl. The cDNAs and the oligo were annealed by incubation at 65°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 100 µl MyOne Streptavidin C1 beads and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 µl 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 µl water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.,,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 supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 µl 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 µl 2x SSC. The RNA was eluted from the beads first with 100 µl 10 mM HEPES pH 7.5 at 65°C for 3 min and then second with 100 µl water at 65°C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 µl water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/µl T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23°C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 µl water. The ligated RNA was mixed with 0.6 µl 100 µM reverse transcription primer KXS037 in a total volume of 12 µl  incubated at 65°C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 µl reaction containing 1x First Strand Buffer  500 µM dNTPs  5 mM dithiothreitol  1 unit/µl SUPERase•In  and 200 units SuperScript III Thermo Fisher  18080044 at 50°C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min  followed by neutralization with 36.7 µl 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer's suggested protocol for 10–15 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3ʹ adapter KXS013 was used for 3ʹ end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 µl. The cDNAs and the oligo were annealed by incubation at 65°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 100 µl MyOne Streptavidin C1 beads and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 µl 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 µl water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.,,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,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 supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 µl 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 µl 2x SSC. The RNA was eluted from the beads first with 100 µl 10 mM HEPES pH 7.5 at 65°C for 3 min and then second with 100 µl water at 65°C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 µl water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/µl T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23°C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 µl water. The ligated RNA was mixed with 0.6 µl 100 µM reverse transcription primer KXS037 in a total volume of 12 µl  incubated at 65°C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 µl reaction containing 1x First Strand Buffer  500 µM dNTPs  5 mM dithiothreitol  1 unit/µl SUPERase•In  and 200 units SuperScript III Thermo Fisher  18080044 at 50°C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min  followed by neutralization with 36.7 µl 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer’s suggested protocol for 10–15 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3ʹ adapter KXS013 was used for 3ʹ end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 µl. The cDNAs and the oligo were annealed by incubation at 65°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 100 µl MyOne Streptavidin C1 beads and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 µl 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 µl water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.,,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 supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 µl 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 µl 2x SSC. The RNA was eluted from the beads first with 100 µl 10 mM HEPES pH 7.5 at 65°C for 3 min and then second with 100 µl water at 65°C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 µl water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/µl T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23°C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 µl water. The ligated RNA was mixed with 0.6 µl 100 µM reverse transcription primer KXS037 in a total volume of 12 µl  incubated at 65°C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 µl reaction containing 1x First Strand Buffer  500 µM dNTPs  5 mM dithiothreitol  1 unit/µl SUPERase•In  and 200 units SuperScript III Thermo Fisher  18080044 at 50°C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min  followed by neutralization with 36.7 µl 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer's suggested protocol for 10–15 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3ʹ adapter KXS013 was used for 3ʹ end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 µl. The cDNAs and the oligo were annealed by incubation at 65°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 100 µl MyOne Streptavidin C1 beads and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 µl 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 µl water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.,,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,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 supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 µl 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 µl 2x SSC. The RNA was eluted from the beads first with 100 µl 10 mM HEPES pH 7.5 at 65°C for 3 min and then second with 100 µl water at 65°C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 µl water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/µl T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23°C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 µl water. The ligated RNA was mixed with 0.6 µl 100 µM reverse transcription primer KXS037 in a total volume of 12 µl  incubated at 65°C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 µl reaction containing 1x First Strand Buffer  500 µM dNTPs  5 mM dithiothreitol  1 unit/µl SUPERase•In  and 200 units SuperScript III Thermo Fisher  18080044 at 50°C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min  followed by neutralization with 36.7 µl 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer’s suggested protocol for 10–15 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3ʹ adapter KXS013 was used for 3ʹ end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 µl. The cDNAs and the oligo were annealed by incubation at 65°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 100 µl MyOne Streptavidin C1 beads and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 µl 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 µl water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.,,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 supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 µl 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 µl 2x SSC. The RNA was eluted from the beads first with 100 µl 10 mM HEPES pH 7.5 at 65°C for 3 min and then second with 100 µl water at 65°C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 µl water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/µl T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23°C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 µl water. The ligated RNA was mixed with 0.6 µl 100 µM reverse transcription primer KXS037 in a total volume of 12 µl  incubated at 65°C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 µl reaction containing 1x First Strand Buffer  500 µM dNTPs  5 mM dithiothreitol  1 unit/µl SUPERase•In  and 200 units SuperScript III Thermo Fisher  18080044 at 50°C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min  followed by neutralization with 36.7 µl 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer's suggested protocol for 10–15 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3ʹ adapter KXS013 was used for 3ʹ end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 µl. The cDNAs and the oligo were annealed by incubation at 65°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 100 µl MyOne Streptavidin C1 beads and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 µl 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 µl water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.,,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,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 supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 µl 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 µl 2x SSC. The RNA was eluted from the beads first with 100 µl 10 mM HEPES pH 7.5 at 65°C for 3 min and then second with 100 µl water at 65°C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 µl water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/µl T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23°C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 µl water. The ligated RNA was mixed with 0.6 µl 100 µM reverse transcription primer KXS037 in a total volume of 12 µl  incubated at 65°C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 µl reaction containing 1x First Strand Buffer  500 µM dNTPs  5 mM dithiothreitol  1 unit/µl SUPERase•In  and 200 units SuperScript III Thermo Fisher  18080044 at 50°C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min  followed by neutralization with 36.7 µl 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer’s suggested protocol for 10–15 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3ʹ adapter KXS013 was used for 3ʹ end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 µl. The cDNAs and the oligo were annealed by incubation at 65°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 100 µl MyOne Streptavidin C1 beads and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 µl 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 µl water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.,,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 supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 µl 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 µl 2x SSC. The RNA was eluted from the beads first with 100 µl 10 mM HEPES pH 7.5 at 65°C for 3 min and then second with 100 µl water at 65°C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 µl water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/µl T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23°C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 µl water. The ligated RNA was mixed with 0.6 µl 100 µM reverse transcription primer KXS037 in a total volume of 12 µl  incubated at 65°C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 µl reaction containing 1x First Strand Buffer  500 µM dNTPs  5 mM dithiothreitol  1 unit/µl SUPERase•In  and 200 units SuperScript III Thermo Fisher  18080044 at 50°C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min  followed by neutralization with 36.7 µl 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer's suggested protocol for 10–15 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3ʹ adapter KXS013 was used for 3ʹ end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 µl. The cDNAs and the oligo were annealed by incubation at 65°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 100 µl MyOne Streptavidin C1 beads and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 µl 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 µl water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.,,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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,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 barcode  gene names  and raw counts for all cells Supplementary files format and content: Metadata metadata.csv: library identity  cell barcode  and associated metadata for all cells   time point  treatment  replicate  annotated cell state  total number of counts  etc Library strategy: inDrops v3 scRNA seq",whole embryo,Zebrafish embryos were arrested for cell cycle using two complementary approaches. S phase cell cycle arrest was induced using a cocktail of drugs – 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,SRR29411985,SRX24925451,SRS21630866,SRP513930,PRJNA1124008,Cell state transitions are decoupled from cell division during early embryo development [II],GSE269848,Other,Paper abstract: As tissues develop  cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation  we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However  in the absence of cell division  differentiation slows down in some cell types  and cells exhibit global stress responses. While differentiation is robust to blocking cell division  the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: Design of experiment for this particular dataset: Forty tails from zebrafish embryos at 24  38 hpf and 48 hpf were collected. Cells were dissociated from these tails and multi seq tags were added to the cells for each time point. Cells from all time points were pooled and single cell sequencing was performed using Indrops. Four gene expression GEX libraries were prepared for transcriptomic information and four multiseq tag libraries TAG were prepared for reading out the multiseq tags. We provide the raw data .fastq files  processed data   both raw counts.tsv.gz and filtered by total UMI counts filtered.h5ad files and the entire filtered data post removing doublets as all data.h5ad. We also provide counts for multi seq tags example  TAG 1.counts.csv. Information about pooling libraries and library indices is provided in Pooling Samples.xlsx. Only 24 hpf data was used for Kukreja et. al. 2024 and hence the cell state information we provide in the all data.h5ad is only for 24 hpf.,,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 files format and content: Raw counts for multi seq tags csv.gz: cell barcode  tag sequence  and raw counts for all cells Supplementary files format and content: Filtered barcodes filtered.h5ad: cell barcode  and associated metadata for all cells Library strategy: inDrops v3 scRNA seq",embryo tail,,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,SRR29411986,SRX24925451,SRS21630866,SRP513930,PRJNA1124008,Cell state transitions are decoupled from cell division during early embryo development [II],GSE269848,Other,Paper abstract: As tissues develop  cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation  we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. However  in the absence of cell division  differentiation slows down in some cell types  and cells exhibit global stress responses. While differentiation is robust to blocking cell division  the proportions of cells across cell states are not but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo wide perturbations with single cell RNA sequencing to uncover the role of common biological processes across multiple tissues. Overall design: Design of experiment for this particular dataset: Forty tails from zebrafish embryos at 24  38 hpf and 48 hpf were collected. Cells were dissociated from these tails and multi seq tags were added to the cells for each time point. Cells from all time points were pooled and single cell sequencing was performed using Indrops. Four gene expression GEX libraries were prepared for transcriptomic information and four multiseq tag libraries TAG were prepared for reading out the multiseq tags. We provide the raw data .fastq files  processed data   both raw counts.tsv.gz and filtered by total UMI counts filtered.h5ad files and the entire filtered data post removing doublets as all data.h5ad. We also provide counts for multi seq tags example  TAG 1.counts.csv. Information about pooling libraries and library indices is provided in Pooling Samples.xlsx. Only 24 hpf data was used for Kukreja et. al. 2024 and hence the cell state information we provide in the all data.h5ad is only for 24 hpf.,,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 files format and content: Raw counts for multi seq tags csv.gz: cell barcode  tag sequence  and raw counts for all cells Supplementary files format and content: Filtered barcodes filtered.h5ad: cell barcode  and associated metadata for all cells Library strategy: inDrops v3 scRNA seq",embryo tail,,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,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,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,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,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,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 8 in 2 mL of media in 2 mL glass vials from xxx hpf ttwo xxx hpf. RNA was collected at at 48 hpf. Exposures  collections and extractions were conducted over two days. Targeted transcriptomics was preformed using the TempO seq platform.,,,,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 single sequencing library 5 µL each into a 1.5mL Eppendorf tube the pooled purified libraries were sequenced using one lane of the NovaseqX 10 B flow cell BioSpyder Technologies  Inc.  Carlsbad  CA  United States.,,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 single sequencing library 5 µL each into a 1.5mL Eppendorf tube the pooled purified libraries were sequenced using one lane of the NovaseqX 10 B flow cell BioSpyder Technologies  Inc.  Carlsbad  CA  United States.,,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,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 8 in 2 mL of media in 2 mL glass vials from xxx hpf ttwo xxx hpf. RNA was collected at at 48 hpf. Exposures  collections and extractions were conducted over two days. Targeted transcriptomics was preformed using the TempO seq platform.,,,,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 single sequencing library 5 µL each into a 1.5mL Eppendorf tube the pooled purified libraries were sequenced using one lane of the NovaseqX 10 B flow cell BioSpyder Technologies  Inc.  Carlsbad  CA  United States.,,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 single sequencing library 5 µL each into a 1.5mL Eppendorf tube the pooled purified libraries were sequenced using one lane of the NovaseqX 10 B flow cell BioSpyder Technologies  Inc.  Carlsbad  CA  United States.,,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,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 8 in 2 mL of media in 2 mL glass vials from xxx hpf ttwo xxx hpf. RNA was collected at at 48 hpf. Exposures  collections and extractions were conducted over two days. Targeted transcriptomics was preformed using the TempO seq platform.,,,,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 single sequencing library 5 µL each into a 1.5mL Eppendorf tube the pooled purified libraries were sequenced using one lane of the NovaseqX 10 B flow cell BioSpyder Technologies  Inc.  Carlsbad  CA  United States.,,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 single sequencing library 5 µL each into a 1.5mL Eppendorf tube the pooled purified libraries were sequenced using one lane of the NovaseqX 10 B flow cell BioSpyder Technologies  Inc.  Carlsbad  CA  United States.,,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,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 8 in 2 mL of media in 2 mL glass vials from xxx hpf ttwo xxx hpf. RNA was collected at at 48 hpf. Exposures  collections and extractions were conducted over two days. Targeted transcriptomics was preformed using the TempO seq platform.,,,,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 single sequencing library 5 µL each into a 1.5mL Eppendorf tube the pooled purified libraries were sequenced using one lane of the NovaseqX 10 B flow cell BioSpyder Technologies  Inc.  Carlsbad  CA  United States.,,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 single sequencing library 5 µL each into a 1.5mL Eppendorf tube the pooled purified libraries were sequenced using one lane of the NovaseqX 10 B flow cell BioSpyder Technologies  Inc.  Carlsbad  CA  United States.,,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,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 8 in 2 mL of media in 2 mL glass vials from xxx hpf ttwo xxx hpf. RNA was collected at at 48 hpf. Exposures  collections and extractions were conducted over two days. Targeted transcriptomics was preformed using the TempO seq platform.,,,,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 single sequencing library 5 µL each into a 1.5mL Eppendorf tube the pooled purified libraries were sequenced using one lane of the NovaseqX 10 B flow cell BioSpyder Technologies  Inc.  Carlsbad  CA  United States.,,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 single sequencing library 5 µL each into a 1.5mL Eppendorf tube the pooled purified libraries were sequenced using one lane of the NovaseqX 10 B flow cell BioSpyder Technologies  Inc.  Carlsbad  CA  United States.,,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,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 8 in 2 mL of media in 2 mL glass vials from xxx hpf ttwo xxx hpf. RNA was collected at at 48 hpf. Exposures  collections and extractions were conducted over two days. Targeted transcriptomics was preformed using the TempO seq platform.,,,,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 single sequencing library 5 µL each into a 1.5mL Eppendorf tube the pooled purified libraries were sequenced using one lane of the NovaseqX 10 B flow cell BioSpyder Technologies  Inc.  Carlsbad  CA  United States.,,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 single sequencing library 5 µL each into a 1.5mL Eppendorf tube the pooled purified libraries were sequenced using one lane of the NovaseqX 10 B flow cell BioSpyder Technologies  Inc.  Carlsbad  CA  United States.,,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,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 8 in 2 mL of media in 2 mL glass vials from xxx hpf ttwo xxx hpf. RNA was collected at at 48 hpf. Exposures  collections and extractions were conducted over two days. Targeted transcriptomics was preformed using the TempO seq platform.,,,,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 single sequencing library 5 µL each into a 1.5mL Eppendorf tube the pooled purified libraries were sequenced using one lane of the NovaseqX 10 B flow cell BioSpyder Technologies  Inc.  Carlsbad  CA  United States.,,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 single sequencing library 5 µL each into a 1.5mL Eppendorf tube the pooled purified libraries were sequenced using one lane of the NovaseqX 10 B flow cell BioSpyder Technologies  Inc.  Carlsbad  CA  United States.,,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,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 8 in 2 mL of media in 2 mL glass vials from xxx hpf ttwo xxx hpf. RNA was collected at at 48 hpf. Exposures  collections and extractions were conducted over two days. Targeted transcriptomics was preformed using the TempO seq platform.,,,,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 single sequencing library 5 µL each into a 1.5mL Eppendorf tube the pooled purified libraries were sequenced using one lane of the NovaseqX 10 B flow cell BioSpyder Technologies  Inc.  Carlsbad  CA  United States.,,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 single sequencing library 5 µL each into a 1.5mL Eppendorf tube the pooled purified libraries were sequenced using one lane of the NovaseqX 10 B flow cell BioSpyder Technologies  Inc.  Carlsbad  CA  United States.,,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,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 8 in 2 mL of media in 2 mL glass vials from xxx hpf ttwo xxx hpf. RNA was collected at at 48 hpf. Exposures  collections and extractions were conducted over two days. Targeted transcriptomics was preformed using the TempO seq platform.,,,,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 single sequencing library 5 µL each into a 1.5mL Eppendorf tube the pooled purified libraries were sequenced using one lane of the NovaseqX 10 B flow cell BioSpyder Technologies  Inc.  Carlsbad  CA  United States.,,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 single sequencing library 5 µL each into a 1.5mL Eppendorf tube the pooled purified libraries were sequenced using one lane of the NovaseqX 10 B flow cell BioSpyder Technologies  Inc.  Carlsbad  CA  United States.,,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,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 8 in 2 mL of media in 2 mL glass vials from xxx hpf ttwo xxx hpf. RNA was collected at at 48 hpf. Exposures  collections and extractions were conducted over two days. Targeted transcriptomics was preformed using the TempO seq platform.,,,,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 single sequencing library 5 µL each into a 1.5mL Eppendorf tube the pooled purified libraries were sequenced using one lane of the NovaseqX 10 B flow cell BioSpyder Technologies  Inc.  Carlsbad  CA  United States.,,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 single sequencing library 5 µL each into a 1.5mL Eppendorf tube the pooled purified libraries were sequenced using one lane of the NovaseqX 10 B flow cell BioSpyder Technologies  Inc.  Carlsbad  CA  United States.,,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,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 8 in 2 mL of media in 2 mL glass vials from xxx hpf ttwo xxx hpf. RNA was collected at at 48 hpf. Exposures  collections and extractions were conducted over two days. Targeted transcriptomics was preformed using the TempO seq platform.,,,,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 single sequencing library 5 µL each into a 1.5mL Eppendorf tube the pooled purified libraries were sequenced using one lane of the NovaseqX 10 B flow cell BioSpyder Technologies  Inc.  Carlsbad  CA  United States.,,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 single sequencing library 5 µL each into a 1.5mL Eppendorf tube the pooled purified libraries were sequenced using one lane of the NovaseqX 10 B flow cell BioSpyder Technologies  Inc.  Carlsbad  CA  United States.,,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,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 8 in 2 mL of media in 2 mL glass vials from xxx hpf ttwo xxx hpf. RNA was collected at at 48 hpf. Exposures  collections and extractions were conducted over two days. Targeted transcriptomics was preformed using the TempO seq platform.,,,,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 single sequencing library 5 µL each into a 1.5mL Eppendorf tube the pooled purified libraries were sequenced using one lane of the NovaseqX 10 B flow cell BioSpyder Technologies  Inc.  Carlsbad  CA  United States.,,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 single sequencing library 5 µL each into a 1.5mL Eppendorf tube the pooled purified libraries were sequenced using one lane of the NovaseqX 10 B flow cell BioSpyder Technologies  Inc.  Carlsbad  CA  United States.,,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,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 8 in 2 mL of media in 2 mL glass vials from xxx hpf ttwo xxx hpf. RNA was collected at at 48 hpf. Exposures  collections and extractions were conducted over two days. Targeted transcriptomics was preformed using the TempO seq platform.,,,,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 single sequencing library 5 µL each into a 1.5mL Eppendorf tube the pooled purified libraries were sequenced using one lane of the NovaseqX 10 B flow cell BioSpyder Technologies  Inc.  Carlsbad  CA  United States.,,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 single sequencing library 5 µL each into a 1.5mL Eppendorf tube the pooled purified libraries were sequenced using one lane of the NovaseqX 10 B flow cell BioSpyder Technologies  Inc.  Carlsbad  CA  United States.,,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,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 8 in 2 mL of media in 2 mL glass vials from xxx hpf ttwo xxx hpf. RNA was collected at at 48 hpf. Exposures  collections and extractions were conducted over two days. Targeted transcriptomics was preformed using the TempO seq platform.,,,,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 single sequencing library 5 µL each into a 1.5mL Eppendorf tube the pooled purified libraries were sequenced using one lane of the NovaseqX 10 B flow cell BioSpyder Technologies  Inc.  Carlsbad  CA  United States.,,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 single sequencing library 5 µL each into a 1.5mL Eppendorf tube the pooled purified libraries were sequenced using one lane of the NovaseqX 10 B flow cell BioSpyder Technologies  Inc.  Carlsbad  CA  United States.,,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,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 8 in 2 mL of media in 2 mL glass vials from xxx hpf ttwo xxx hpf. RNA was collected at at 48 hpf. Exposures  collections and extractions were conducted over two days. Targeted transcriptomics was preformed using the TempO seq platform.,,,,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 single sequencing library 5 µL each into a 1.5mL Eppendorf tube the pooled purified libraries were sequenced using one lane of the NovaseqX 10 B flow cell BioSpyder Technologies  Inc.  Carlsbad  CA  United States.,,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 single sequencing library 5 µL each into a 1.5mL Eppendorf tube the pooled purified libraries were sequenced using one lane of the NovaseqX 10 B flow cell BioSpyder Technologies  Inc.  Carlsbad  CA  United States.,,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,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 8 in 2 mL of media in 2 mL glass vials from xxx hpf ttwo xxx hpf. RNA was collected at at 48 hpf. Exposures  collections and extractions were conducted over two days. Targeted transcriptomics was preformed using the TempO seq platform.,,,,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 single sequencing library 5 µL each into a 1.5mL Eppendorf tube the pooled purified libraries were sequenced using one lane of the NovaseqX 10 B flow cell BioSpyder Technologies  Inc.  Carlsbad  CA  United States.,,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 single sequencing library 5 µL each into a 1.5mL Eppendorf tube the pooled purified libraries were sequenced using one lane of the NovaseqX 10 B flow cell BioSpyder Technologies  Inc.  Carlsbad  CA  United States.,,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,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 8 in 2 mL of media in 2 mL glass vials from xxx hpf ttwo xxx hpf. RNA was collected at at 48 hpf. Exposures  collections and extractions were conducted over two days. Targeted transcriptomics was preformed using the TempO seq platform.,,,,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 single sequencing library 5 µL each into a 1.5mL Eppendorf tube the pooled purified libraries were sequenced using one lane of the NovaseqX 10 B flow cell BioSpyder Technologies  Inc.  Carlsbad  CA  United States.,,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 single sequencing library 5 µL each into a 1.5mL Eppendorf tube the pooled purified libraries were sequenced using one lane of the NovaseqX 10 B flow cell BioSpyder Technologies  Inc.  Carlsbad  CA  United States.,,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,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 8 in 2 mL of media in 2 mL glass vials from xxx hpf ttwo xxx hpf. RNA was collected at at 48 hpf. Exposures  collections and extractions were conducted over two days. Targeted transcriptomics was preformed using the TempO seq platform.,,,,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 single sequencing library 5 µL each into a 1.5mL Eppendorf tube the pooled purified libraries were sequenced using one lane of the NovaseqX 10 B flow cell BioSpyder Technologies  Inc.  Carlsbad  CA  United States.,,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 single sequencing library 5 µL each into a 1.5mL Eppendorf tube the pooled purified libraries were sequenced using one lane of the NovaseqX 10 B flow cell BioSpyder Technologies  Inc.  Carlsbad  CA  United States.,,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,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 8 in 2 mL of media in 2 mL glass vials from xxx hpf ttwo xxx hpf. RNA was collected at at 48 hpf. Exposures  collections and extractions were conducted over two days. Targeted transcriptomics was preformed using the TempO seq platform.,,,,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 single sequencing library 5 µL each into a 1.5mL Eppendorf tube the pooled purified libraries were sequenced using one lane of the NovaseqX 10 B flow cell BioSpyder Technologies  Inc.  Carlsbad  CA  United States.,,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 single sequencing library 5 µL each into a 1.5mL Eppendorf tube the pooled purified libraries were sequenced using one lane of the NovaseqX 10 B flow cell BioSpyder Technologies  Inc.  Carlsbad  CA  United States.,,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,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 8 in 2 mL of media in 2 mL glass vials from xxx hpf ttwo xxx hpf. RNA was collected at at 48 hpf. Exposures  collections and extractions were conducted over two days. Targeted transcriptomics was preformed using the TempO seq platform.,,,,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 single sequencing library 5 µL each into a 1.5mL Eppendorf tube the pooled purified libraries were sequenced using one lane of the NovaseqX 10 B flow cell BioSpyder Technologies  Inc.  Carlsbad  CA  United States.,,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 single sequencing library 5 µL each into a 1.5mL Eppendorf tube the pooled purified libraries were sequenced using one lane of the NovaseqX 10 B flow cell BioSpyder Technologies  Inc.  Carlsbad  CA  United States.,,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,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 8 in 2 mL of media in 2 mL glass vials from xxx hpf ttwo xxx hpf. RNA was collected at at 48 hpf. Exposures  collections and extractions were conducted over two days. Targeted transcriptomics was preformed using the TempO seq platform.,,,,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 single sequencing library 5 µL each into a 1.5mL Eppendorf tube the pooled purified libraries were sequenced using one lane of the NovaseqX 10 B flow cell BioSpyder Technologies  Inc.  Carlsbad  CA  United States.,,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 single sequencing library 5 µL each into a 1.5mL Eppendorf tube the pooled purified libraries were sequenced using one lane of the NovaseqX 10 B flow cell BioSpyder Technologies  Inc.  Carlsbad  CA  United States.,,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,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 8 in 2 mL of media in 2 mL glass vials from xxx hpf ttwo xxx hpf. RNA was collected at at 48 hpf. Exposures  collections and extractions were conducted over two days. Targeted transcriptomics was preformed using the TempO seq platform.,,,,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 single sequencing library 5 µL each into a 1.5mL Eppendorf tube the pooled purified libraries were sequenced using one lane of the NovaseqX 10 B flow cell BioSpyder Technologies  Inc.  Carlsbad  CA  United States.,,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 single sequencing library 5 µL each into a 1.5mL Eppendorf tube the pooled purified libraries were sequenced using one lane of the NovaseqX 10 B flow cell BioSpyder Technologies  Inc.  Carlsbad  CA  United States.,,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,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 8 in 2 mL of media in 2 mL glass vials from xxx hpf ttwo xxx hpf. RNA was collected at at 48 hpf. Exposures  collections and extractions were conducted over two days. Targeted transcriptomics was preformed using the TempO seq platform.,,,,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 single sequencing library 5 µL each into a 1.5mL Eppendorf tube the pooled purified libraries were sequenced using one lane of the NovaseqX 10 B flow cell BioSpyder Technologies  Inc.  Carlsbad  CA  United States.,,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 single sequencing library 5 µL each into a 1.5mL Eppendorf tube the pooled purified libraries were sequenced using one lane of the NovaseqX 10 B flow cell BioSpyder Technologies  Inc.  Carlsbad  CA  United States.,,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,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 8 in 2 mL of media in 2 mL glass vials from xxx hpf ttwo xxx hpf. RNA was collected at at 48 hpf. Exposures  collections and extractions were conducted over two days. Targeted transcriptomics was preformed using the TempO seq platform.,,,,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 single sequencing library 5 µL each into a 1.5mL Eppendorf tube the pooled purified libraries were sequenced using one lane of the NovaseqX 10 B flow cell BioSpyder Technologies  Inc.  Carlsbad  CA  United States.,,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 single sequencing library 5 µL each into a 1.5mL Eppendorf tube the pooled purified libraries were sequenced using one lane of the NovaseqX 10 B flow cell BioSpyder Technologies  Inc.  Carlsbad  CA  United States.,,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,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 8 in 2 mL of media in 2 mL glass vials from xxx hpf ttwo xxx hpf. RNA was collected at at 48 hpf. Exposures  collections and extractions were conducted over two days. Targeted transcriptomics was preformed using the TempO seq platform.,,,,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 single sequencing library 5 µL each into a 1.5mL Eppendorf tube the pooled purified libraries were sequenced using one lane of the NovaseqX 10 B flow cell BioSpyder Technologies  Inc.  Carlsbad  CA  United States.,,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 single sequencing library 5 µL each into a 1.5mL Eppendorf tube the pooled purified libraries were sequenced using one lane of the NovaseqX 10 B flow cell BioSpyder Technologies  Inc.  Carlsbad  CA  United States.,,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