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
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
25188,SRR25670735,SRX21396039,SRS18636197,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 1024cell PAL seq v4,GSM7716868,,source name:embryo|tissue:embryo|treatment:N1|geo loc name:missing|collection date:missing,Fish embryo mRNA 1024cell 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,GSM7716868,GSM7716868: Fish embryo mRNA 1024cell PAL seq v4; Danio rerio; OTHER,GSM7716868 r1,GSM7716868,1,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_1024cell_PAL_seq_v4_rep1_raw_read1.fastq.gz Fish_embryo_mRNA_1024cell_PAL_seq_v4_rep1_raw_read2.fastq.gz,fastq fastq,2191900794.0,7139742.0,GSM7716868 r1,0:52 1:255,A:571954354;C:551162617;G:572445400;T:490238669;N:6099754,52,255,,,571954354,551162617,572445400,490238669,6099754,SRX21396039,SRS18636197,SRA1694849,Whitehead Institute,Whitehead Institute,2,0.0011,0.43387,6e-05,0.01058,0.99864,0.99971,0.74576,1.0,52,255,T,B,mate1 technical by mapping diff,illumina,hiseq_era,unknown,poly_a,unknown,bulk,bulk,bulk,,United States,2023-08-17,Zygote,Embryo,Embryo Imprecise,All anatomical structures
25189,SRR25670736,SRX21396039,SRS18636197,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 1024cell PAL seq v4,GSM7716868,,source name:embryo|tissue:embryo|treatment:N1|geo loc name:missing|collection date:missing,Fish embryo mRNA 1024cell 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,GSM7716868,GSM7716868: Fish embryo mRNA 1024cell PAL seq v4; Danio rerio; OTHER,GSM7716868 r1,GSM7716868,1,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_1024cell_PAL_seq_v4_rep2_raw_read1.fastq.gz Fish_embryo_mRNA_1024cell_PAL_seq_v4_rep2_raw_read2.fastq.gz,fastq fastq,3840723193.0,12510499.0,GSM7716868 r2,0:52 1:255,A:985340216;C:991311706;G:1034487140;T:821820974;N:7763157,52,255,,,985340216,991311706,1034487140,821820974,7763157,SRX21396039,SRS18636197,SRA1694849,Whitehead Institute,Whitehead Institute,2,0.0021,0.0,0.00026,0.0,0.99859,1.0,0.71022,,52,255,T,T,mates < 9% mapping rate,illumina,hiseq_era,unknown,poly_a,unknown,bulk,bulk,bulk,,United States,2023-08-17,Zygote,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
25194,SRR25670741,SRX21396036,SRS18636194,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 1cell PAL seq v4,GSM7716865,,source name:embryo|tissue:embryo|treatment:N1|geo loc name:missing|collection date:missing,Fish embryo mRNA 1cell 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,GSM7716865,GSM7716865: Fish embryo mRNA 1cell PAL seq v4; Danio rerio; OTHER,GSM7716865 r1,GSM7716865,1,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_1cell_PAL_seq_v4_rep1_raw_read1.fastq.gz Fish_embryo_mRNA_1cell_PAL_seq_v4_rep1_raw_read2.fastq.gz,fastq fastq,2050143851.0,6677993.0,GSM7716865 r1,0:52 1:255,A:536391564;C:527126186;G:557802929;T:422990166;N:5833006,52,255,,,536391564,527126186,557802929,422990166,5833006,SRX21396036,SRS18636194,SRA1694849,Whitehead Institute,Whitehead Institute,2,0.00016,0.46479,4e-05,0.01408,0.99979,0.99969,0.54545,1.0,52,255,T,B,mate1 technical by mapping diff,illumina,hiseq_era,unknown,poly_a,unknown,bulk,bulk,bulk,,United States,2023-08-17,Zygote,Embryo,Embryo Imprecise,All anatomical structures
25195,SRR25670742,SRX21396036,SRS18636194,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 1cell PAL seq v4,GSM7716865,,source name:embryo|tissue:embryo|treatment:N1|geo loc name:missing|collection date:missing,Fish embryo mRNA 1cell 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,GSM7716865,GSM7716865: Fish embryo mRNA 1cell PAL seq v4; Danio rerio; OTHER,GSM7716865 r1,GSM7716865,1,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_1cell_PAL_seq_v4_rep2_raw_read1.fastq.gz Fish_embryo_mRNA_1cell_PAL_seq_v4_rep2_raw_read2.fastq.gz,fastq fastq,3406093776.0,11094768.0,GSM7716865 r2,0:52 1:255,A:868054279;C:890446070;G:945289336;T:695324057;N:6980034,52,255,,,868054279,890446070,945289336,695324057,6980034,SRX21396036,SRS18636194,SRA1694849,Whitehead Institute,Whitehead Institute,2,0.00049,0.0,0.00014,0.0,0.99939,1.0,0.58974,,52,255,T,T,mates < 9% mapping rate,illumina,hiseq_era,unknown,poly_a,unknown,bulk,bulk,bulk,,United States,2023-08-17,Zygote,Embryo,Embryo Imprecise,All anatomical structures
36373,SRR499871,SRX148961,SRS334490,SRP013309,PRJNA167302,Transcriptomic profiles of zebrafish embryos exposed to silver nanoparticles  bulk and ions using HT SuperSAGE in a Illumina GA2 platform,GSE38125,Other,Silver nanoparticles cause toxicity in exposed organisms and are an environmental health concern. The mechanisms of silver nanoparticle toxicity  however  remain unclear. We examined the effects of exposure to silver in nano   bulk  and ionic forms on zebrafish embryos Danio rerio using a Next Generation Sequencing approach in an Illumina platform High Throughput SuperSAGE. Significant alterations in gene expression were found for all treatments and many of the gene pathways affected  most notably those associated with oxidative phosphorylation and protein synthesis  overlapped strongly between the three treatments indicating similar mechanisms of toxicity for the three forms of silver studied. Changes in oxidative phosphorylation indicated a down regulation of this pathway at 24h of exposure  but with a recovery at 48h. This finding was consistent with a dose dependent decrease in oxygen consumption at 24h  but not at 48h  following exposure to silver ions. Overall  our data provide support for the hypothesis that the toxicity caused by silver nanoparticles is principally associated with bioavailable silver ions in exposed zebrafish embryos. These findings are important in the evaluation of the risk that silver particles may pose to exposed vertebrate organisms. Overall design: mRNA profiles of whole zebrafish embryos at 24 hpf and 48 hpf exposed to silver in nano  bulk and ionic forms were generated by deep sequencing using HT SuperSAGE Illumina GA2.,,pubmed:23758687,,Dre 48h silver ions,GSM935123,,source name:Dre 48h silver ions|strain:wild type WIK strain|tissue:whole embryos|developmental stage:48 hpf|treatment:silver ions 0.25 µg/L of silver nitrate|barcode:GCGA,Dre 48h silver ions,FASTQ/A Barcode splitter from the FASTX Toolkit http://hannonlab.cshl.edu/fastx toolkit/download.html was used to separate the samples from each lane using the 4 base barcode. FASTQ to FASTA from the FASTX Toolkit was used to convert the fastq files to fasta files. FASTQ/A trimmer from the FASTX Toolkit was used to remove the barcodes the first 4 bases from the sequence. A Perl script was used to remove all bases post the last occurence of CATG NlaIII restriction site  used for sequence tag preparation in each of the sequences FASTX collapser was used to collapse the sequences and calculate the frequency of unique sequence tags unitags in each library Supplementary files format and content: tabulated text files include frequency of all unitags in the treatment library,Dre 48h silver ions,Stock solutions for Ag NP  Ag Bulk and silver nitrate were made up in ultrapure water  and sonicated for 1 h to ensure dispersal of the particles. Exposures were conducted in glass chambers  at 28+/  1°C with a 12h light: dark photoperiod. Immediately prior to the start of the exposures  glass chambers received 400 mL of ISO water  prepared according to OECD guidelines for zebrafish embryo experiments http://www.oecd.org/  containing 5 µg/L of 10nm Ag NP  5  µg/L of Ag Bulk or 0.25  µg/L of silver nitrate. A control chamber was set up containing water alone.  Solutions were replaced every 12h during the exposure period  and dead embryos were removed at the times of replacement of the exposure water. At 24h and 48h  3 pools of 50 embryos were removed from each exposure tank  immediately frozen in liquid nitrogen and stored at  80°C for analysis of gene expression. The experiment was terminated at 48 hpf.,High throughput HT SuperSAGE libraries were prepared as described in Matsumura et al 2010  PLoS ONE 58:e12010. Samples were multiplexed on each lane of the Illumina flow cell.,Adult WIK zebrafish were kept in the aquarium facilites at the University of Exeter according to the protocols described in Paull et al.  2008 Aquat Toxicol. 872:115 26. Fish were allowed to breed naturally and eggs were collected in glass egg chambers  approximately 1 hpf. Eggs were then cleaned and unfertilised embryos were removed prior to the exposures.,strain:wild type WIK strain|tissue:whole embryos|developmental stage:48 hpf|treatment:silver ions 0.25 µg/L of silver nitrate|barcode:GCGA,GSM935123,GSM935123: Dre 48h silver ions; Danio rerio; OTHER,GSM935123 1,GSM935123: Dre 48h silver ions,1,,GEO Accession:GSM935123,OTHER,TRANSCRIPTOMIC,other,SINGLE,ILLUMINA,Illumina Genome Analyzer II,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>36</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,SRP013309,,,Dre_48h_ag.gz,fastq,85514364.0,2375399.0,GSM935123 r1,0:36,A:18075876;C:24481350;G:23393114;T:19494907;N:69117,36,,,,18075876,24481350,23393114,19494907,69117,SRX148961,SRS334490,SRA053074,GEO,"van Aerle Lab, Biosciences, College of Life and Environmental Sciences, University of Exeter",1,0.11101,,0.00946,,0.96224,,0.6991,,36,,B,,usable mapping rate,illumina,early_illumina,unknown,other,unknown,bulk,bulk,bulk,,United Kingdom,2012-05-22,Hatching,Embryo,Whole Organism,All anatomical structures
36374,SRR499870,SRX148960,SRS334489,SRP013309,PRJNA167302,Transcriptomic profiles of zebrafish embryos exposed to silver nanoparticles  bulk and ions using HT SuperSAGE in a Illumina GA2 platform,GSE38125,Other,Silver nanoparticles cause toxicity in exposed organisms and are an environmental health concern. The mechanisms of silver nanoparticle toxicity  however  remain unclear. We examined the effects of exposure to silver in nano   bulk  and ionic forms on zebrafish embryos Danio rerio using a Next Generation Sequencing approach in an Illumina platform High Throughput SuperSAGE. Significant alterations in gene expression were found for all treatments and many of the gene pathways affected  most notably those associated with oxidative phosphorylation and protein synthesis  overlapped strongly between the three treatments indicating similar mechanisms of toxicity for the three forms of silver studied. Changes in oxidative phosphorylation indicated a down regulation of this pathway at 24h of exposure  but with a recovery at 48h. This finding was consistent with a dose dependent decrease in oxygen consumption at 24h  but not at 48h  following exposure to silver ions. Overall  our data provide support for the hypothesis that the toxicity caused by silver nanoparticles is principally associated with bioavailable silver ions in exposed zebrafish embryos. These findings are important in the evaluation of the risk that silver particles may pose to exposed vertebrate organisms. Overall design: mRNA profiles of whole zebrafish embryos at 24 hpf and 48 hpf exposed to silver in nano  bulk and ionic forms were generated by deep sequencing using HT SuperSAGE Illumina GA2.,,pubmed:23758687,,Dre 48h silver bulk,GSM935122,,source name:Dre 48h silver bulk|strain:wild type WIK strain|tissue:whole embryos|developmental stage:48 hpf|treatment:silver bulk 0.6 1.6µm; 5 µg/L|barcode:GCGC,Dre 48h silver bulk,FASTQ/A Barcode splitter from the FASTX Toolkit http://hannonlab.cshl.edu/fastx toolkit/download.html was used to separate the samples from each lane using the 4 base barcode. FASTQ to FASTA from the FASTX Toolkit was used to convert the fastq files to fasta files. FASTQ/A trimmer from the FASTX Toolkit was used to remove the barcodes the first 4 bases from the sequence. A Perl script was used to remove all bases post the last occurence of CATG NlaIII restriction site  used for sequence tag preparation in each of the sequences FASTX collapser was used to collapse the sequences and calculate the frequency of unique sequence tags unitags in each library Supplementary files format and content: tabulated text files include frequency of all unitags in the treatment library,Dre 48h silver bulk,Stock solutions for Ag NP  Ag Bulk and silver nitrate were made up in ultrapure water  and sonicated for 1 h to ensure dispersal of the particles. Exposures were conducted in glass chambers  at 28+/  1°C with a 12h light: dark photoperiod. Immediately prior to the start of the exposures  glass chambers received 400 mL of ISO water  prepared according to OECD guidelines for zebrafish embryo experiments http://www.oecd.org/  containing 5 µg/L of 10nm Ag NP  5  µg/L of Ag Bulk or 0.25  µg/L of silver nitrate. A control chamber was set up containing water alone.  Solutions were replaced every 12h during the exposure period  and dead embryos were removed at the times of replacement of the exposure water. At 24h and 48h  3 pools of 50 embryos were removed from each exposure tank  immediately frozen in liquid nitrogen and stored at  80°C for analysis of gene expression. The experiment was terminated at 48 hpf.,High throughput HT SuperSAGE libraries were prepared as described in Matsumura et al 2010  PLoS ONE 58:e12010. Samples were multiplexed on each lane of the Illumina flow cell.,Adult WIK zebrafish were kept in the aquarium facilites at the University of Exeter according to the protocols described in Paull et al.  2008 Aquat Toxicol. 872:115 26. Fish were allowed to breed naturally and eggs were collected in glass egg chambers  approximately 1 hpf. Eggs were then cleaned and unfertilised embryos were removed prior to the exposures.,strain:wild type WIK strain|tissue:whole embryos|developmental stage:48 hpf|treatment:silver bulk 0.6 1.6µm; 5 µg/L|barcode:GCGC,GSM935122,GSM935122: Dre 48h silver bulk; Danio rerio; OTHER,GSM935122 1,GSM935122: Dre 48h silver bulk,1,,GEO Accession:GSM935122,OTHER,TRANSCRIPTOMIC,other,SINGLE,ILLUMINA,Illumina Genome Analyzer II,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>36</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,SRP013309,,,Dre_48h_bulk.gz,fastq,95750712.0,2659742.0,GSM935122 r1,0:36,A:17624901;C:29735122;G:26376026;T:21938121;N:76542,36,,,,17624901,29735122,26376026,21938121,76542,SRX148960,SRS334489,SRA053074,GEO,"van Aerle Lab, Biosciences, College of Life and Environmental Sciences, University of Exeter",1,0.18319,,0.01693,,0.94391,,0.57004,,36,,B,,usable mapping rate,illumina,early_illumina,unknown,other,unknown,bulk,bulk,bulk,,United Kingdom,2012-05-22,Hatching,Embryo,Whole Organism,All anatomical structures
36375,SRR499869,SRX148959,SRS334488,SRP013309,PRJNA167302,Transcriptomic profiles of zebrafish embryos exposed to silver nanoparticles  bulk and ions using HT SuperSAGE in a Illumina GA2 platform,GSE38125,Other,Silver nanoparticles cause toxicity in exposed organisms and are an environmental health concern. The mechanisms of silver nanoparticle toxicity  however  remain unclear. We examined the effects of exposure to silver in nano   bulk  and ionic forms on zebrafish embryos Danio rerio using a Next Generation Sequencing approach in an Illumina platform High Throughput SuperSAGE. Significant alterations in gene expression were found for all treatments and many of the gene pathways affected  most notably those associated with oxidative phosphorylation and protein synthesis  overlapped strongly between the three treatments indicating similar mechanisms of toxicity for the three forms of silver studied. Changes in oxidative phosphorylation indicated a down regulation of this pathway at 24h of exposure  but with a recovery at 48h. This finding was consistent with a dose dependent decrease in oxygen consumption at 24h  but not at 48h  following exposure to silver ions. Overall  our data provide support for the hypothesis that the toxicity caused by silver nanoparticles is principally associated with bioavailable silver ions in exposed zebrafish embryos. These findings are important in the evaluation of the risk that silver particles may pose to exposed vertebrate organisms. Overall design: mRNA profiles of whole zebrafish embryos at 24 hpf and 48 hpf exposed to silver in nano  bulk and ionic forms were generated by deep sequencing using HT SuperSAGE Illumina GA2.,,pubmed:23758687,,Dre 48h silver NP,GSM935121,,source name:Dre 48h silver NP|strain:wild type WIK strain|tissue:whole embryos|developmental stage:48 hpf|treatment:silver nanoparticles 10nm; 5µg/L|barcode:GCTG,Dre 48h silver NP,FASTQ/A Barcode splitter from the FASTX Toolkit http://hannonlab.cshl.edu/fastx toolkit/download.html was used to separate the samples from each lane using the 4 base barcode. FASTQ to FASTA from the FASTX Toolkit was used to convert the fastq files to fasta files. FASTQ/A trimmer from the FASTX Toolkit was used to remove the barcodes the first 4 bases from the sequence. A Perl script was used to remove all bases post the last occurence of CATG NlaIII restriction site  used for sequence tag preparation in each of the sequences FASTX collapser was used to collapse the sequences and calculate the frequency of unique sequence tags unitags in each library Supplementary files format and content: tabulated text files include frequency of all unitags in the treatment library,Dre 48h silver NP,Stock solutions for Ag NP  Ag Bulk and silver nitrate were made up in ultrapure water  and sonicated for 1 h to ensure dispersal of the particles. Exposures were conducted in glass chambers  at 28+/  1°C with a 12h light: dark photoperiod. Immediately prior to the start of the exposures  glass chambers received 400 mL of ISO water  prepared according to OECD guidelines for zebrafish embryo experiments http://www.oecd.org/  containing 5 µg/L of 10nm Ag NP  5  µg/L of Ag Bulk or 0.25  µg/L of silver nitrate. A control chamber was set up containing water alone.  Solutions were replaced every 12h during the exposure period  and dead embryos were removed at the times of replacement of the exposure water. At 24h and 48h  3 pools of 50 embryos were removed from each exposure tank  immediately frozen in liquid nitrogen and stored at  80°C for analysis of gene expression. The experiment was terminated at 48 hpf.,High throughput HT SuperSAGE libraries were prepared as described in Matsumura et al 2010  PLoS ONE 58:e12010. Samples were multiplexed on each lane of the Illumina flow cell.,Adult WIK zebrafish were kept in the aquarium facilites at the University of Exeter according to the protocols described in Paull et al.  2008 Aquat Toxicol. 872:115 26. Fish were allowed to breed naturally and eggs were collected in glass egg chambers  approximately 1 hpf. Eggs were then cleaned and unfertilised embryos were removed prior to the exposures.,strain:wild type WIK strain|tissue:whole embryos|developmental stage:48 hpf|treatment:silver nanoparticles 10nm; 5µg/L|barcode:GCTG,GSM935121,GSM935121: Dre 48h silver NP; Danio rerio; OTHER,GSM935121 1,GSM935121: Dre 48h silver NP,1,,GEO Accession:GSM935121,OTHER,TRANSCRIPTOMIC,other,SINGLE,ILLUMINA,Illumina Genome Analyzer II,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>36</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,SRP013309,,,Dre_48h_NP.gz,fastq,52931304.0,1470314.0,GSM935121 r1,0:36,A:10003539;C:15279729;G:14487570;T:13116768;N:43698,36,,,,10003539,15279729,14487570,13116768,43698,SRX148959,SRS334488,SRA053074,GEO,"van Aerle Lab, Biosciences, College of Life and Environmental Sciences, University of Exeter",1,0.14206,,0.01226,,0.95477,,0.58652,,36,,B,,usable mapping rate,illumina,early_illumina,unknown,other,unknown,bulk,bulk,bulk,,United Kingdom,2012-05-22,Hatching,Embryo,Whole Organism,All anatomical structures
36376,SRR499868,SRX148958,SRS334487,SRP013309,PRJNA167302,Transcriptomic profiles of zebrafish embryos exposed to silver nanoparticles  bulk and ions using HT SuperSAGE in a Illumina GA2 platform,GSE38125,Other,Silver nanoparticles cause toxicity in exposed organisms and are an environmental health concern. The mechanisms of silver nanoparticle toxicity  however  remain unclear. We examined the effects of exposure to silver in nano   bulk  and ionic forms on zebrafish embryos Danio rerio using a Next Generation Sequencing approach in an Illumina platform High Throughput SuperSAGE. Significant alterations in gene expression were found for all treatments and many of the gene pathways affected  most notably those associated with oxidative phosphorylation and protein synthesis  overlapped strongly between the three treatments indicating similar mechanisms of toxicity for the three forms of silver studied. Changes in oxidative phosphorylation indicated a down regulation of this pathway at 24h of exposure  but with a recovery at 48h. This finding was consistent with a dose dependent decrease in oxygen consumption at 24h  but not at 48h  following exposure to silver ions. Overall  our data provide support for the hypothesis that the toxicity caused by silver nanoparticles is principally associated with bioavailable silver ions in exposed zebrafish embryos. These findings are important in the evaluation of the risk that silver particles may pose to exposed vertebrate organisms. Overall design: mRNA profiles of whole zebrafish embryos at 24 hpf and 48 hpf exposed to silver in nano  bulk and ionic forms were generated by deep sequencing using HT SuperSAGE Illumina GA2.,,pubmed:23758687,,Dre 48h control,GSM935120,,source name:Dre 48h control|strain:wild type WIK strain|tissue:whole embryos|developmental stage:48 hpf|treatment:control|barcode:GCTT,Dre 48h control,FASTQ/A Barcode splitter from the FASTX Toolkit http://hannonlab.cshl.edu/fastx toolkit/download.html was used to separate the samples from each lane using the 4 base barcode. FASTQ to FASTA from the FASTX Toolkit was used to convert the fastq files to fasta files. FASTQ/A trimmer from the FASTX Toolkit was used to remove the barcodes the first 4 bases from the sequence. A Perl script was used to remove all bases post the last occurence of CATG NlaIII restriction site  used for sequence tag preparation in each of the sequences FASTX collapser was used to collapse the sequences and calculate the frequency of unique sequence tags unitags in each library Supplementary files format and content: tabulated text files include frequency of all unitags in the treatment library,Dre 48h control,Stock solutions for Ag NP  Ag Bulk and silver nitrate were made up in ultrapure water  and sonicated for 1 h to ensure dispersal of the particles. Exposures were conducted in glass chambers  at 28+/  1°C with a 12h light: dark photoperiod. Immediately prior to the start of the exposures  glass chambers received 400 mL of ISO water  prepared according to OECD guidelines for zebrafish embryo experiments http://www.oecd.org/  containing 5 µg/L of 10nm Ag NP  5  µg/L of Ag Bulk or 0.25  µg/L of silver nitrate. A control chamber was set up containing water alone.  Solutions were replaced every 12h during the exposure period  and dead embryos were removed at the times of replacement of the exposure water. At 24h and 48h  3 pools of 50 embryos were removed from each exposure tank  immediately frozen in liquid nitrogen and stored at  80°C for analysis of gene expression. The experiment was terminated at 48 hpf.,High throughput HT SuperSAGE libraries were prepared as described in Matsumura et al 2010  PLoS ONE 58:e12010. Samples were multiplexed on each lane of the Illumina flow cell.,Adult WIK zebrafish were kept in the aquarium facilites at the University of Exeter according to the protocols described in Paull et al.  2008 Aquat Toxicol. 872:115 26. Fish were allowed to breed naturally and eggs were collected in glass egg chambers  approximately 1 hpf. Eggs were then cleaned and unfertilised embryos were removed prior to the exposures.,strain:wild type WIK strain|tissue:whole embryos|developmental stage:48 hpf|treatment:control|barcode:GCTT,GSM935120,GSM935120: Dre 48h control; Danio rerio; OTHER,GSM935120 1,GSM935120: Dre 48h control,1,,GEO Accession:GSM935120,OTHER,TRANSCRIPTOMIC,other,SINGLE,ILLUMINA,Illumina Genome Analyzer II,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>36</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,SRP013309,,,Dre_48h_control.gz,fastq,75389652.0,2094157.0,GSM935120 r1,0:36,A:13252992;C:21927091;G:18790968;T:21232585;N:186016,36,,,,13252992,21927091,18790968,21232585,186016,SRX148958,SRS334487,SRA053074,GEO,"van Aerle Lab, Biosciences, College of Life and Environmental Sciences, University of Exeter",1,0.12359,,0.01182,,0.95929,,0.56028,,36,,B,,usable mapping rate,illumina,early_illumina,unknown,other,unknown,bulk,bulk,bulk,,United Kingdom,2012-05-22,Hatching,Embryo,Whole Organism,All anatomical structures
36377,SRR499867,SRX148957,SRS334486,SRP013309,PRJNA167302,Transcriptomic profiles of zebrafish embryos exposed to silver nanoparticles  bulk and ions using HT SuperSAGE in a Illumina GA2 platform,GSE38125,Other,Silver nanoparticles cause toxicity in exposed organisms and are an environmental health concern. The mechanisms of silver nanoparticle toxicity  however  remain unclear. We examined the effects of exposure to silver in nano   bulk  and ionic forms on zebrafish embryos Danio rerio using a Next Generation Sequencing approach in an Illumina platform High Throughput SuperSAGE. Significant alterations in gene expression were found for all treatments and many of the gene pathways affected  most notably those associated with oxidative phosphorylation and protein synthesis  overlapped strongly between the three treatments indicating similar mechanisms of toxicity for the three forms of silver studied. Changes in oxidative phosphorylation indicated a down regulation of this pathway at 24h of exposure  but with a recovery at 48h. This finding was consistent with a dose dependent decrease in oxygen consumption at 24h  but not at 48h  following exposure to silver ions. Overall  our data provide support for the hypothesis that the toxicity caused by silver nanoparticles is principally associated with bioavailable silver ions in exposed zebrafish embryos. These findings are important in the evaluation of the risk that silver particles may pose to exposed vertebrate organisms. Overall design: mRNA profiles of whole zebrafish embryos at 24 hpf and 48 hpf exposed to silver in nano  bulk and ionic forms were generated by deep sequencing using HT SuperSAGE Illumina GA2.,,pubmed:23758687,,Dre 24h silver ions,GSM935119,,source name:Dre 24h silver ions|strain:wild type WIK strain|tissue:whole embryos|developmental stage:24 hpf|treatment:silver ions 0.25 µg/L of silver nitrate|barcode:GCTA,Dre 24h silver ions,FASTQ/A Barcode splitter from the FASTX Toolkit http://hannonlab.cshl.edu/fastx toolkit/download.html was used to separate the samples from each lane using the 4 base barcode. FASTQ to FASTA from the FASTX Toolkit was used to convert the fastq files to fasta files. FASTQ/A trimmer from the FASTX Toolkit was used to remove the barcodes the first 4 bases from the sequence. A Perl script was used to remove all bases post the last occurence of CATG NlaIII restriction site  used for sequence tag preparation in each of the sequences FASTX collapser was used to collapse the sequences and calculate the frequency of unique sequence tags unitags in each library Supplementary files format and content: tabulated text files include frequency of all unitags in the treatment library,Dre 24h silver ions,Stock solutions for Ag NP  Ag Bulk and silver nitrate were made up in ultrapure water  and sonicated for 1 h to ensure dispersal of the particles. Exposures were conducted in glass chambers  at 28+/  1°C with a 12h light: dark photoperiod. Immediately prior to the start of the exposures  glass chambers received 400 mL of ISO water  prepared according to OECD guidelines for zebrafish embryo experiments http://www.oecd.org/  containing 5 µg/L of 10nm Ag NP  5  µg/L of Ag Bulk or 0.25  µg/L of silver nitrate. A control chamber was set up containing water alone.  Solutions were replaced every 12h during the exposure period  and dead embryos were removed at the times of replacement of the exposure water. At 24h and 48h  3 pools of 50 embryos were removed from each exposure tank  immediately frozen in liquid nitrogen and stored at  80°C for analysis of gene expression. The experiment was terminated at 48 hpf.,High throughput HT SuperSAGE libraries were prepared as described in Matsumura et al 2010  PLoS ONE 58:e12010. Samples were multiplexed on each lane of the Illumina flow cell.,Adult WIK zebrafish were kept in the aquarium facilites at the University of Exeter according to the protocols described in Paull et al.  2008 Aquat Toxicol. 872:115 26. Fish were allowed to breed naturally and eggs were collected in glass egg chambers  approximately 1 hpf. Eggs were then cleaned and unfertilised embryos were removed prior to the exposures.,strain:wild type WIK strain|tissue:whole embryos|developmental stage:24 hpf|treatment:silver ions 0.25 µg/L of silver nitrate|barcode:GCTA,GSM935119,GSM935119: Dre 24h silver ions; Danio rerio; OTHER,GSM935119 1,GSM935119: Dre 24h silver ions,1,,GEO Accession:GSM935119,OTHER,TRANSCRIPTOMIC,other,SINGLE,ILLUMINA,Illumina Genome Analyzer II,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>36</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,SRP013309,,,Dre_24h_ag.gz,fastq,57745404.0,1604039.0,GSM935119 r1,0:36,A:11280863;C:17795869;G:15444087;T:13175334;N:49251,36,,,,11280863,17795869,15444087,13175334,49251,SRX148957,SRS334486,SRA053074,GEO,"van Aerle Lab, Biosciences, College of Life and Environmental Sciences, University of Exeter",1,0.10143,,0.00773,,0.96435,,0.60432,,36,,B,,usable mapping rate,illumina,early_illumina,unknown,other,unknown,bulk,bulk,bulk,,United Kingdom,2012-05-22,Pharyngula,Embryo,Whole Organism,All anatomical structures
36378,SRR499866,SRX148956,SRS334485,SRP013309,PRJNA167302,Transcriptomic profiles of zebrafish embryos exposed to silver nanoparticles  bulk and ions using HT SuperSAGE in a Illumina GA2 platform,GSE38125,Other,Silver nanoparticles cause toxicity in exposed organisms and are an environmental health concern. The mechanisms of silver nanoparticle toxicity  however  remain unclear. We examined the effects of exposure to silver in nano   bulk  and ionic forms on zebrafish embryos Danio rerio using a Next Generation Sequencing approach in an Illumina platform High Throughput SuperSAGE. Significant alterations in gene expression were found for all treatments and many of the gene pathways affected  most notably those associated with oxidative phosphorylation and protein synthesis  overlapped strongly between the three treatments indicating similar mechanisms of toxicity for the three forms of silver studied. Changes in oxidative phosphorylation indicated a down regulation of this pathway at 24h of exposure  but with a recovery at 48h. This finding was consistent with a dose dependent decrease in oxygen consumption at 24h  but not at 48h  following exposure to silver ions. Overall  our data provide support for the hypothesis that the toxicity caused by silver nanoparticles is principally associated with bioavailable silver ions in exposed zebrafish embryos. These findings are important in the evaluation of the risk that silver particles may pose to exposed vertebrate organisms. Overall design: mRNA profiles of whole zebrafish embryos at 24 hpf and 48 hpf exposed to silver in nano  bulk and ionic forms were generated by deep sequencing using HT SuperSAGE Illumina GA2.,,pubmed:23758687,,Dre 24h silver bulk,GSM935118,,source name:Dre 24h silver bulk|strain:wild type WIK strain|tissue:whole embryos|developmental stage:24 hpf|treatment:silver bulk 0.6 1.6µm; 5 µg/L|barcode:GCTC,Dre 24h silver bulk,FASTQ/A Barcode splitter from the FASTX Toolkit http://hannonlab.cshl.edu/fastx toolkit/download.html was used to separate the samples from each lane using the 4 base barcode. FASTQ to FASTA from the FASTX Toolkit was used to convert the fastq files to fasta files. FASTQ/A trimmer from the FASTX Toolkit was used to remove the barcodes the first 4 bases from the sequence. A Perl script was used to remove all bases post the last occurence of CATG NlaIII restriction site  used for sequence tag preparation in each of the sequences FASTX collapser was used to collapse the sequences and calculate the frequency of unique sequence tags unitags in each library Supplementary files format and content: tabulated text files include frequency of all unitags in the treatment library,Dre 24h silver bulk,Stock solutions for Ag NP  Ag Bulk and silver nitrate were made up in ultrapure water  and sonicated for 1 h to ensure dispersal of the particles. Exposures were conducted in glass chambers  at 28+/  1°C with a 12h light: dark photoperiod. Immediately prior to the start of the exposures  glass chambers received 400 mL of ISO water  prepared according to OECD guidelines for zebrafish embryo experiments http://www.oecd.org/  containing 5 µg/L of 10nm Ag NP  5  µg/L of Ag Bulk or 0.25  µg/L of silver nitrate. A control chamber was set up containing water alone.  Solutions were replaced every 12h during the exposure period  and dead embryos were removed at the times of replacement of the exposure water. At 24h and 48h  3 pools of 50 embryos were removed from each exposure tank  immediately frozen in liquid nitrogen and stored at  80°C for analysis of gene expression. The experiment was terminated at 48 hpf.,High throughput HT SuperSAGE libraries were prepared as described in Matsumura et al 2010  PLoS ONE 58:e12010. Samples were multiplexed on each lane of the Illumina flow cell.,Adult WIK zebrafish were kept in the aquarium facilites at the University of Exeter according to the protocols described in Paull et al.  2008 Aquat Toxicol. 872:115 26. Fish were allowed to breed naturally and eggs were collected in glass egg chambers  approximately 1 hpf. Eggs were then cleaned and unfertilised embryos were removed prior to the exposures.,strain:wild type WIK strain|tissue:whole embryos|developmental stage:24 hpf|treatment:silver bulk 0.6 1.6µm; 5 µg/L|barcode:GCTC,GSM935118,GSM935118: Dre 24h silver bulk; Danio rerio; OTHER,GSM935118 1,GSM935118: Dre 24h silver bulk,1,,GEO Accession:GSM935118,OTHER,TRANSCRIPTOMIC,other,SINGLE,ILLUMINA,Illumina Genome Analyzer II,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>36</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,SRP013309,,,Dre_24h_bulk.gz,fastq,53296632.0,1480462.0,GSM935118 r1,0:36,A:8986044;C:17367907;G:13631579;T:13181001;N:130101,36,,,,8986044,17367907,13631579,13181001,130101,SRX148956,SRS334485,SRA053074,GEO,"van Aerle Lab, Biosciences, College of Life and Environmental Sciences, University of Exeter",1,0.078,,0.00708,,0.96877,,0.4609,,36,,B,,usable mapping rate,illumina,early_illumina,unknown,other,unknown,bulk,bulk,bulk,,United Kingdom,2012-05-22,Pharyngula,Embryo,Whole Organism,All anatomical structures
36379,SRR499865,SRX148955,SRS334484,SRP013309,PRJNA167302,Transcriptomic profiles of zebrafish embryos exposed to silver nanoparticles  bulk and ions using HT SuperSAGE in a Illumina GA2 platform,GSE38125,Other,Silver nanoparticles cause toxicity in exposed organisms and are an environmental health concern. The mechanisms of silver nanoparticle toxicity  however  remain unclear. We examined the effects of exposure to silver in nano   bulk  and ionic forms on zebrafish embryos Danio rerio using a Next Generation Sequencing approach in an Illumina platform High Throughput SuperSAGE. Significant alterations in gene expression were found for all treatments and many of the gene pathways affected  most notably those associated with oxidative phosphorylation and protein synthesis  overlapped strongly between the three treatments indicating similar mechanisms of toxicity for the three forms of silver studied. Changes in oxidative phosphorylation indicated a down regulation of this pathway at 24h of exposure  but with a recovery at 48h. This finding was consistent with a dose dependent decrease in oxygen consumption at 24h  but not at 48h  following exposure to silver ions. Overall  our data provide support for the hypothesis that the toxicity caused by silver nanoparticles is principally associated with bioavailable silver ions in exposed zebrafish embryos. These findings are important in the evaluation of the risk that silver particles may pose to exposed vertebrate organisms. Overall design: mRNA profiles of whole zebrafish embryos at 24 hpf and 48 hpf exposed to silver in nano  bulk and ionic forms were generated by deep sequencing using HT SuperSAGE Illumina GA2.,,pubmed:23758687,,Dre 24h silver NP,GSM935117,,source name:Dre 24h silver NP|strain:wild type WIK strain|tissue:whole embryos|developmental stage:24 hpf|treatment:silver nanoparticles 10nm; 5µg/L|barcode:GCAG,Dre 24h silver NP,FASTQ/A Barcode splitter from the FASTX Toolkit http://hannonlab.cshl.edu/fastx toolkit/download.html was used to separate the samples from each lane using the 4 base barcode. FASTQ to FASTA from the FASTX Toolkit was used to convert the fastq files to fasta files. FASTQ/A trimmer from the FASTX Toolkit was used to remove the barcodes the first 4 bases from the sequence. A Perl script was used to remove all bases post the last occurence of CATG NlaIII restriction site  used for sequence tag preparation in each of the sequences FASTX collapser was used to collapse the sequences and calculate the frequency of unique sequence tags unitags in each library Supplementary files format and content: tabulated text files include frequency of all unitags in the treatment library,Dre 24h silver NP,Stock solutions for Ag NP  Ag Bulk and silver nitrate were made up in ultrapure water  and sonicated for 1 h to ensure dispersal of the particles. Exposures were conducted in glass chambers  at 28+/  1°C with a 12h light: dark photoperiod. Immediately prior to the start of the exposures  glass chambers received 400 mL of ISO water  prepared according to OECD guidelines for zebrafish embryo experiments http://www.oecd.org/  containing 5 µg/L of 10nm Ag NP  5  µg/L of Ag Bulk or 0.25  µg/L of silver nitrate. A control chamber was set up containing water alone.  Solutions were replaced every 12h during the exposure period  and dead embryos were removed at the times of replacement of the exposure water. At 24h and 48h  3 pools of 50 embryos were removed from each exposure tank  immediately frozen in liquid nitrogen and stored at  80°C for analysis of gene expression. The experiment was terminated at 48 hpf.,High throughput HT SuperSAGE libraries were prepared as described in Matsumura et al 2010  PLoS ONE 58:e12010. Samples were multiplexed on each lane of the Illumina flow cell.,Adult WIK zebrafish were kept in the aquarium facilites at the University of Exeter according to the protocols described in Paull et al.  2008 Aquat Toxicol. 872:115 26. Fish were allowed to breed naturally and eggs were collected in glass egg chambers  approximately 1 hpf. Eggs were then cleaned and unfertilised embryos were removed prior to the exposures.,strain:wild type WIK strain|tissue:whole embryos|developmental stage:24 hpf|treatment:silver nanoparticles 10nm; 5µg/L|barcode:GCAG,GSM935117,GSM935117: Dre 24h silver NP; Danio rerio; OTHER,GSM935117 1,GSM935117: Dre 24h silver NP,1,,GEO Accession:GSM935117,OTHER,TRANSCRIPTOMIC,other,SINGLE,ILLUMINA,Illumina Genome Analyzer II,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>36</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,SRP013309,,,Dre_24h_NP.gz,fastq,103827096.0,2884086.0,GSM935117 r1,0:36,A:22437552;C:29017806;G:28385826;T:23907583;N:78329,36,,,,22437552,29017806,28385826,23907583,78329,SRX148955,SRS334484,SRA053074,GEO,"van Aerle Lab, Biosciences, College of Life and Environmental Sciences, University of Exeter",1,0.16321,,0.01606,,0.94631,,0.45783,,36,,B,,usable mapping rate,illumina,early_illumina,unknown,other,unknown,bulk,bulk,bulk,,United Kingdom,2012-05-22,Pharyngula,Embryo,Whole Organism,All anatomical structures
36380,SRR499864,SRX148954,SRS334483,SRP013309,PRJNA167302,Transcriptomic profiles of zebrafish embryos exposed to silver nanoparticles  bulk and ions using HT SuperSAGE in a Illumina GA2 platform,GSE38125,Other,Silver nanoparticles cause toxicity in exposed organisms and are an environmental health concern. The mechanisms of silver nanoparticle toxicity  however  remain unclear. We examined the effects of exposure to silver in nano   bulk  and ionic forms on zebrafish embryos Danio rerio using a Next Generation Sequencing approach in an Illumina platform High Throughput SuperSAGE. Significant alterations in gene expression were found for all treatments and many of the gene pathways affected  most notably those associated with oxidative phosphorylation and protein synthesis  overlapped strongly between the three treatments indicating similar mechanisms of toxicity for the three forms of silver studied. Changes in oxidative phosphorylation indicated a down regulation of this pathway at 24h of exposure  but with a recovery at 48h. This finding was consistent with a dose dependent decrease in oxygen consumption at 24h  but not at 48h  following exposure to silver ions. Overall  our data provide support for the hypothesis that the toxicity caused by silver nanoparticles is principally associated with bioavailable silver ions in exposed zebrafish embryos. These findings are important in the evaluation of the risk that silver particles may pose to exposed vertebrate organisms. Overall design: mRNA profiles of whole zebrafish embryos at 24 hpf and 48 hpf exposed to silver in nano  bulk and ionic forms were generated by deep sequencing using HT SuperSAGE Illumina GA2.,,pubmed:23758687,,Dre 24h control,GSM935116,,source name:Dre 24h control|strain:wild type WIK strain|tissue:whole embryos|developmental stage:24 hpf|treatment:control|barcode:GCAT,Dre 24h control,FASTQ/A Barcode splitter from the FASTX Toolkit http://hannonlab.cshl.edu/fastx toolkit/download.html was used to separate the samples from each lane using the 4 base barcode. FASTQ to FASTA from the FASTX Toolkit was used to convert the fastq files to fasta files. FASTQ/A trimmer from the FASTX Toolkit was used to remove the barcodes the first 4 bases from the sequence. A Perl script was used to remove all bases post the last occurence of CATG NlaIII restriction site  used for sequence tag preparation in each of the sequences FASTX collapser was used to collapse the sequences and calculate the frequency of unique sequence tags unitags in each library Supplementary files format and content: tabulated text files include frequency of all unitags in the treatment library,Dre 24h control,Stock solutions for Ag NP  Ag Bulk and silver nitrate were made up in ultrapure water  and sonicated for 1 h to ensure dispersal of the particles. Exposures were conducted in glass chambers  at 28+/  1°C with a 12h light: dark photoperiod. Immediately prior to the start of the exposures  glass chambers received 400 mL of ISO water  prepared according to OECD guidelines for zebrafish embryo experiments http://www.oecd.org/  containing 5 µg/L of 10nm Ag NP  5  µg/L of Ag Bulk or 0.25  µg/L of silver nitrate. A control chamber was set up containing water alone.  Solutions were replaced every 12h during the exposure period  and dead embryos were removed at the times of replacement of the exposure water. At 24h and 48h  3 pools of 50 embryos were removed from each exposure tank  immediately frozen in liquid nitrogen and stored at  80°C for analysis of gene expression. The experiment was terminated at 48 hpf.,High throughput HT SuperSAGE libraries were prepared as described in Matsumura et al 2010  PLoS ONE 58:e12010. Samples were multiplexed on each lane of the Illumina flow cell.,Adult WIK zebrafish were kept in the aquarium facilites at the University of Exeter according to the protocols described in Paull et al.  2008 Aquat Toxicol. 872:115 26. Fish were allowed to breed naturally and eggs were collected in glass egg chambers  approximately 1 hpf. Eggs were then cleaned and unfertilised embryos were removed prior to the exposures.,strain:wild type WIK strain|tissue:whole embryos|developmental stage:24 hpf|treatment:control|barcode:GCAT,GSM935116,GSM935116: Dre 24h control; Danio rerio; OTHER,GSM935116 1,GSM935116: Dre 24h control,1,,GEO Accession:GSM935116,OTHER,TRANSCRIPTOMIC,other,SINGLE,ILLUMINA,Illumina Genome Analyzer II,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>36</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,SRP013309,,,Dre_24h_control.gz,fastq,85032468.0,2362013.0,GSM935116 r1,0:36,A:16524315;C:24822030;G:21632275;T:21837611;N:216237,36,,,,16524315,24822030,21632275,21837611,216237,SRX148954,SRS334483,SRA053074,GEO,"van Aerle Lab, Biosciences, College of Life and Environmental Sciences, University of Exeter",1,0.13983,,0.01175,,0.95966,,0.52467,,36,,B,,usable mapping rate,illumina,early_illumina,unknown,other,unknown,bulk,bulk,bulk,,United Kingdom,2012-05-22,Pharyngula,Embryo,Whole Organism,All anatomical structures
41521,SRR5004953,SRX2336787,SRS1790184,SRP092907,PRJNA352850,Transcriptomics analysis of gene expressions  m6A enrichment levels and ythdf2 binding targets in control and mettl3 or ythdf2 morphants zebrafish embryos,GSE89655,Other,RNA was isolated from control and mettl3 or ythdf2 morphants zebrafish cells using the TRIzol Invitrogen reagent by following the company manual. For all samples the RNA integrity was checked using an Agilent Bioanalyzer 2100. All samples showed a RIN RNA integrity number of higher than 9. Approximately 2.5 µg of total RNA was then used for library preparation using a TruSeq™ RNA Sample Prep Kit v2 Illumina  San Diego  CA  USA according to the manufacturer’s protocol.The libraries were sequenced using HiSeq2500 or HiSeq 3000 Illumina in paired read mode  creating reads with a length of 101 or 151 bp. Sequencing chemistry v2 Illumina was used. Overall design: Examination of gene expressive levels  m6A enrichment levels  m6A single uncleotide sites and ythdf2 binding targets in normal and mettl3 or ythdf2 morphants zebrafish cells,,pubmed:28869969,,mettl3 morphant zebrafish embryos m6A IP rep2,GSM2386182,,source name:zebrafish embryos|genotype/variation:mettl3|cell type:embryos cells|age:28hpf|tissue:trunk|enrichment antibody vendor:Synaptic Systems,mettl3 morphant zebrafish embryos m6A IP rep2,Library strategy: MeRIP seq Reads were aligned to the zv9 genome assembly using TopHat v2.0.9 Genome build: zv9 Supplementary files format and content: m6A peak locus.xls: m6A peaks indentified by macs2 software Supplementary files format and content: reads count HTSeq.xls: Reads count for each gene of all samples,zebrafish embryos,,For MeRIP seq  mRNA was isolated from the trunck region of zebrafish embryos at 28hpf. For IP samples  mRNA was sonicated and subjected to IP using m6A antibody Synaptic Systems  202003. The RNA integrity was checked using an Agilent Bioanalyzer 2100. Approximately 2.5 µg of total RNA was then used for library preparation using a TruSeq™ RNA Sample Prep Kit v2 Illumina  San Diego  CA  USA according to the manufacturer’s protocol.The libraries were sequenced using HiSeq3000 Illumina in paired read mode  creating reads with a length of 101 bp.,,genotype/variation:mettl3|cell type:embryos cells|age:28hpf|tissue:trunk|enrichment antibody vendor:Synaptic Systems,GSM2386182,GSM2386182: mettl3 morphant zebrafish embryos m6A IP rep2; Danio rerio; OTHER,GSM2386182,,1,For MeRIP seq  mRNA was isolated from the trunck region of zebrafish embryos at 28hpf. For IP samples  mRNA was sonicated and subjected to IP using m6A antibody Synaptic Systems  202003. The RNA integrity was checked using an Agilent Bioanalyzer 2100. Approximately 2.5 µg of total RNA was then used for library preparation using a TruSeq™ RNA Sample Prep Kit v2 Illumina  San Diego  CA  USA according to the manufacturer’s protocol.The libraries were sequenced using HiSeq3000 Illumina in paired read mode  creating reads with a length of 101 bp.,GEO Accession:GSM2386182,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,Illumina HiSeq 3000,,SRP092907,,,mettl3-mo_IP_rep2_1.fastq.gz mettl3-mo_IP_rep2_2.fastq.gz,fastq fastq,12246129204.0,60624402.0,GSM2386182 r1,0:101 1:101,A:3253814653;C:2887966071;G:2921051332;T:3179545005;N:3752143,101,101,,,3253814653,2887966071,2921051332,3179545005,3752143,SRX2336787,SRS1790184,SRA491654,GEO,Beijing Institute of Genomics (BIG) of Chinese Academy of Sciences (CAS),2,0.93906,0.94462,0.08073,0.07972,0.69148,0.69394,0.43224,0.48778,101,101,B,B,biological fallback assumption,illumina,hiseq_era,unknown,other,trueseq,bulk,bulk,bulk,,China,2016-11-08,Pharyngula,Embryo,Trunk,Surface Structure
41522,SRR5004952,SRX2336785,SRS1790183,SRP092907,PRJNA352850,Transcriptomics analysis of gene expressions  m6A enrichment levels and ythdf2 binding targets in control and mettl3 or ythdf2 morphants zebrafish embryos,GSE89655,Other,RNA was isolated from control and mettl3 or ythdf2 morphants zebrafish cells using the TRIzol Invitrogen reagent by following the company manual. For all samples the RNA integrity was checked using an Agilent Bioanalyzer 2100. All samples showed a RIN RNA integrity number of higher than 9. Approximately 2.5 µg of total RNA was then used for library preparation using a TruSeq™ RNA Sample Prep Kit v2 Illumina  San Diego  CA  USA according to the manufacturer’s protocol.The libraries were sequenced using HiSeq2500 or HiSeq 3000 Illumina in paired read mode  creating reads with a length of 101 or 151 bp. Sequencing chemistry v2 Illumina was used. Overall design: Examination of gene expressive levels  m6A enrichment levels  m6A single uncleotide sites and ythdf2 binding targets in normal and mettl3 or ythdf2 morphants zebrafish cells,,pubmed:28869969,,mettl3 morphant zebrafish embryos m6A IP rep1,GSM2386181,,source name:zebrafish embryos|genotype/variation:mettl3|cell type:embryos cells|age:28hpf|tissue:trunk|enrichment antibody vendor:Synaptic Systems,mettl3 morphant zebrafish embryos m6A IP rep1,Library strategy: MeRIP seq Reads were aligned to the zv9 genome assembly using TopHat v2.0.9 Genome build: zv9 Supplementary files format and content: m6A peak locus.xls: m6A peaks indentified by macs2 software Supplementary files format and content: reads count HTSeq.xls: Reads count for each gene of all samples,zebrafish embryos,,For MeRIP seq  mRNA was isolated from the trunck region of zebrafish embryos at 28hpf. For IP samples  mRNA was sonicated and subjected to IP using m6A antibody Synaptic Systems  202003. The RNA integrity was checked using an Agilent Bioanalyzer 2100. Approximately 2.5 µg of total RNA was then used for library preparation using a TruSeq™ RNA Sample Prep Kit v2 Illumina  San Diego  CA  USA according to the manufacturer’s protocol.The libraries were sequenced using HiSeq3000 Illumina in paired read mode  creating reads with a length of 151 bp.,,genotype/variation:mettl3|cell type:embryos cells|age:28hpf|tissue:trunk|enrichment antibody vendor:Synaptic Systems,GSM2386181,GSM2386181: mettl3 morphant zebrafish embryos m6A IP rep1; Danio rerio; OTHER,GSM2386181,,1,For MeRIP seq  mRNA was isolated from the trunck region of zebrafish embryos at 28hpf. For IP samples  mRNA was sonicated and subjected to IP using m6A antibody Synaptic Systems  202003. The RNA integrity was checked using an Agilent Bioanalyzer 2100. Approximately 2.5 µg of total RNA was then used for library preparation using a TruSeq™ RNA Sample Prep Kit v2 Illumina  San Diego  CA  USA according to the manufacturer’s protocol.The libraries were sequenced using HiSeq3000 Illumina in paired read mode  creating reads with a length of 151 bp.,GEO Accession:GSM2386181,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,Illumina HiSeq 3000,,SRP092907,,,mettl3-mo_IP_rep1_1.fastq.gz mettl3-mo_IP_rep1_2.fastq.gz,fastq fastq,22767872412.0,75390306.0,GSM2386181 r1,0:151 1:151,A:6356270201;C:5175380564;G:5545741592;T:5686194578;N:4285477,151,151,,,6356270201,5175380564,5545741592,5686194578,4285477,SRX2336785,SRS1790183,SRA491654,GEO,Beijing Institute of Genomics (BIG) of Chinese Academy of Sciences (CAS),2,0.94647,0.95162,0.07314,0.07248,0.70796,0.71021,0.54859,0.54624,151,151,B,B,biological fallback assumption,illumina,hiseq_era,unknown,other,trueseq,bulk,bulk,bulk,,China,2016-11-08,Pharyngula,Embryo,Trunk,Surface Structure
41523,SRR5004951,SRX2336784,SRS1790182,SRP092907,PRJNA352850,Transcriptomics analysis of gene expressions  m6A enrichment levels and ythdf2 binding targets in control and mettl3 or ythdf2 morphants zebrafish embryos,GSE89655,Other,RNA was isolated from control and mettl3 or ythdf2 morphants zebrafish cells using the TRIzol Invitrogen reagent by following the company manual. For all samples the RNA integrity was checked using an Agilent Bioanalyzer 2100. All samples showed a RIN RNA integrity number of higher than 9. Approximately 2.5 µg of total RNA was then used for library preparation using a TruSeq™ RNA Sample Prep Kit v2 Illumina  San Diego  CA  USA according to the manufacturer’s protocol.The libraries were sequenced using HiSeq2500 or HiSeq 3000 Illumina in paired read mode  creating reads with a length of 101 or 151 bp. Sequencing chemistry v2 Illumina was used. Overall design: Examination of gene expressive levels  m6A enrichment levels  m6A single uncleotide sites and ythdf2 binding targets in normal and mettl3 or ythdf2 morphants zebrafish cells,,pubmed:28869969,,control zebrafish embryos input rep2,GSM2386180,,source name:zebrafish embryos|genotype/variation:normal|cell type:embryos cells|age:28hpf|tissue:trunk,control zebrafish embryos input rep2,Library strategy: MeRIP seq Reads were aligned to the zv9 genome assembly using TopHat v2.0.9 Genome build: zv9 Supplementary files format and content: reads count HTSeq.xls: Reads count for each gene of all samples,zebrafish embryos,,For MeRIP seq  mRNA was isolated from the trunck region of zebrafish embryos at 28hpf. For IP samples  mRNA was sonicated and subjected to IP using m6A antibody Synaptic Systems  202003. The RNA integrity was checked using an Agilent Bioanalyzer 2100. Approximately 2.5 µg of total RNA was then used for library preparation using a TruSeq™ RNA Sample Prep Kit v2 Illumina  San Diego  CA  USA according to the manufacturer’s protocol.The libraries were sequenced using HiSeq3000 Illumina in paired read mode  creating reads with a length of 101 bp.,,genotype/variation:normal|cell type:embryos cells|age:28hpf|tissue:trunk,GSM2386180,GSM2386180: control zebrafish embryos input rep2; Danio rerio; OTHER,GSM2386180,,1,For MeRIP seq  mRNA was isolated from the trunck region of zebrafish embryos at 28hpf. For IP samples  mRNA was sonicated and subjected to IP using m6A antibody Synaptic Systems  202003. The RNA integrity was checked using an Agilent Bioanalyzer 2100. Approximately 2.5 µg of total RNA was then used for library preparation using a TruSeq™ RNA Sample Prep Kit v2 Illumina  San Diego  CA  USA according to the manufacturer’s protocol.The libraries were sequenced using HiSeq3000 Illumina in paired read mode  creating reads with a length of 101 bp.,GEO Accession:GSM2386180,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,Illumina HiSeq 3000,,SRP092907,,,control_input_rep2_1.fastq.gz control_input_rep2_2.fastq.gz,fastq fastq,20629042748.0,102123974.0,GSM2386180 r1,0:101 1:101,A:5541548211;C:4810650367;G:4699999124;T:5571342766;N:5502280,101,101,,,5541548211,4810650367,4699999124,5571342766,5502280,SRX2336784,SRS1790182,SRA491654,GEO,Beijing Institute of Genomics (BIG) of Chinese Academy of Sciences (CAS),2,0.94898,0.9511,0.10842,0.10266,0.70465,0.70481,0.4986,0.48915,101,101,B,B,biological fallback assumption,illumina,hiseq_era,unknown,other,trueseq,bulk,bulk,bulk,,China,2016-11-08,Pharyngula,Embryo,Trunk,Surface Structure
41524,SRR5004950,SRX2336783,SRS1790181,SRP092907,PRJNA352850,Transcriptomics analysis of gene expressions  m6A enrichment levels and ythdf2 binding targets in control and mettl3 or ythdf2 morphants zebrafish embryos,GSE89655,Other,RNA was isolated from control and mettl3 or ythdf2 morphants zebrafish cells using the TRIzol Invitrogen reagent by following the company manual. For all samples the RNA integrity was checked using an Agilent Bioanalyzer 2100. All samples showed a RIN RNA integrity number of higher than 9. Approximately 2.5 µg of total RNA was then used for library preparation using a TruSeq™ RNA Sample Prep Kit v2 Illumina  San Diego  CA  USA according to the manufacturer’s protocol.The libraries were sequenced using HiSeq2500 or HiSeq 3000 Illumina in paired read mode  creating reads with a length of 101 or 151 bp. Sequencing chemistry v2 Illumina was used. Overall design: Examination of gene expressive levels  m6A enrichment levels  m6A single uncleotide sites and ythdf2 binding targets in normal and mettl3 or ythdf2 morphants zebrafish cells,,pubmed:28869969,,control zebrafish embryos input rep1,GSM2386179,,source name:zebrafish embryos|genotype/variation:normal|cell type:embryos cells|age:28hpf|tissue:trunk,control zebrafish embryos input rep1,Library strategy: MeRIP seq Reads were aligned to the zv9 genome assembly using TopHat v2.0.9 Genome build: zv9 Supplementary files format and content: reads count HTSeq.xls: Reads count for each gene of all samples,zebrafish embryos,,For MeRIP seq  mRNA was isolated from the trunck region of zebrafish embryos at 28hpf. For IP samples  mRNA was sonicated and subjected to IP using m6A antibody Synaptic Systems  202003. The RNA integrity was checked using an Agilent Bioanalyzer 2100. Approximately 2.5 µg of total RNA was then used for library preparation using a TruSeq™ RNA Sample Prep Kit v2 Illumina  San Diego  CA  USA according to the manufacturer’s protocol.The libraries were sequenced using HiSeq3000 Illumina in paired read mode  creating reads with a length of 151 bp.,,genotype/variation:normal|cell type:embryos cells|age:28hpf|tissue:trunk,GSM2386179,GSM2386179: control zebrafish embryos input rep1; Danio rerio; OTHER,GSM2386179,,1,For MeRIP seq  mRNA was isolated from the trunck region of zebrafish embryos at 28hpf. For IP samples  mRNA was sonicated and subjected to IP using m6A antibody Synaptic Systems  202003. The RNA integrity was checked using an Agilent Bioanalyzer 2100. Approximately 2.5 µg of total RNA was then used for library preparation using a TruSeq™ RNA Sample Prep Kit v2 Illumina  San Diego  CA  USA according to the manufacturer’s protocol.The libraries were sequenced using HiSeq3000 Illumina in paired read mode  creating reads with a length of 151 bp.,GEO Accession:GSM2386179,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,Illumina HiSeq 3000,,SRP092907,,,control_input_rep1_1.fastq.gz control_input_rep1_2.fastq.gz,fastq fastq,27776308060.0,91974530.0,GSM2386179 r1,0:151 1:151,A:7467418896;C:6517753701;G:6333567394;T:7452805069;N:4763000,151,151,,,7467418896,6517753701,6333567394,7452805069,4763000,SRX2336783,SRS1790181,SRA491654,GEO,Beijing Institute of Genomics (BIG) of Chinese Academy of Sciences (CAS),2,0.95616,0.95939,0.10401,0.09776,0.72263,0.73637,0.57906,0.58185,151,151,B,B,biological fallback assumption,illumina,hiseq_era,unknown,other,trueseq,bulk,bulk,bulk,,China,2016-11-08,Pharyngula,Embryo,Trunk,Surface Structure
41525,SRR5004949,SRX2336782,SRS1790180,SRP092907,PRJNA352850,Transcriptomics analysis of gene expressions  m6A enrichment levels and ythdf2 binding targets in control and mettl3 or ythdf2 morphants zebrafish embryos,GSE89655,Other,RNA was isolated from control and mettl3 or ythdf2 morphants zebrafish cells using the TRIzol Invitrogen reagent by following the company manual. For all samples the RNA integrity was checked using an Agilent Bioanalyzer 2100. All samples showed a RIN RNA integrity number of higher than 9. Approximately 2.5 µg of total RNA was then used for library preparation using a TruSeq™ RNA Sample Prep Kit v2 Illumina  San Diego  CA  USA according to the manufacturer’s protocol.The libraries were sequenced using HiSeq2500 or HiSeq 3000 Illumina in paired read mode  creating reads with a length of 101 or 151 bp. Sequencing chemistry v2 Illumina was used. Overall design: Examination of gene expressive levels  m6A enrichment levels  m6A single uncleotide sites and ythdf2 binding targets in normal and mettl3 or ythdf2 morphants zebrafish cells,,pubmed:28869969,,control zebrafish embryos m6A IP rep2,GSM2386178,,source name:zebrafish embryos|genotype/variation:normal|cell type:embryos cells|age:28hpf|tissue:trunk|enrichment antibody vendor:Synaptic Systems,control zebrafish embryos m6A IP rep2,Library strategy: MeRIP seq Reads were aligned to the zv9 genome assembly using TopHat v2.0.9 Genome build: zv9 Supplementary files format and content: m6A peak locus.xls: m6A peaks indentified by macs2 software Supplementary files format and content: reads count HTSeq.xls: Reads count for each gene of all samples,zebrafish embryos,,For MeRIP seq  mRNA was isolated from the trunck region of zebrafish embryos at 28hpf. For IP samples  mRNA was sonicated and subjected to IP using m6A antibody Synaptic Systems  202003. The RNA integrity was checked using an Agilent Bioanalyzer 2100. Approximately 2.5 µg of total RNA was then used for library preparation using a TruSeq™ RNA Sample Prep Kit v2 Illumina  San Diego  CA  USA according to the manufacturer’s protocol.The libraries were sequenced using HiSeq3000 Illumina in paired read mode  creating reads with a length of 101 bp.,,genotype/variation:normal|cell type:embryos cells|age:28hpf|tissue:trunk|enrichment antibody vendor:Synaptic Systems,GSM2386178,GSM2386178: control zebrafish embryos m6A IP rep2; Danio rerio; OTHER,GSM2386178,,1,For MeRIP seq  mRNA was isolated from the trunck region of zebrafish embryos at 28hpf. For IP samples  mRNA was sonicated and subjected to IP using m6A antibody Synaptic Systems  202003. The RNA integrity was checked using an Agilent Bioanalyzer 2100. Approximately 2.5 µg of total RNA was then used for library preparation using a TruSeq™ RNA Sample Prep Kit v2 Illumina  San Diego  CA  USA according to the manufacturer’s protocol.The libraries were sequenced using HiSeq3000 Illumina in paired read mode  creating reads with a length of 101 bp.,GEO Accession:GSM2386178,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,Illumina HiSeq 3000,,SRP092907,,,control_IP_rep2_1.fastq.gz control_IP_rep2_2.fastq.gz,fastq fastq,11899573964.0,58908782.0,GSM2386178 r1,0:101 1:101,A:3146907590;C:2817914269;G:2838718195;T:3092392965;N:3640945,101,101,,,3146907590,2817914269,2838718195,3092392965,3640945,SRX2336782,SRS1790180,SRA491654,GEO,Beijing Institute of Genomics (BIG) of Chinese Academy of Sciences (CAS),2,0.93821,0.94281,0.08361,0.08174,0.69676,0.69816,0.4628,0.46386,101,101,B,B,biological fallback assumption,illumina,hiseq_era,unknown,other,trueseq,bulk,bulk,bulk,,China,2016-11-08,Pharyngula,Embryo,Trunk,Surface Structure
41526,SRR5004948,SRX2336781,SRS1790179,SRP092907,PRJNA352850,Transcriptomics analysis of gene expressions  m6A enrichment levels and ythdf2 binding targets in control and mettl3 or ythdf2 morphants zebrafish embryos,GSE89655,Other,RNA was isolated from control and mettl3 or ythdf2 morphants zebrafish cells using the TRIzol Invitrogen reagent by following the company manual. For all samples the RNA integrity was checked using an Agilent Bioanalyzer 2100. All samples showed a RIN RNA integrity number of higher than 9. Approximately 2.5 µg of total RNA was then used for library preparation using a TruSeq™ RNA Sample Prep Kit v2 Illumina  San Diego  CA  USA according to the manufacturer’s protocol.The libraries were sequenced using HiSeq2500 or HiSeq 3000 Illumina in paired read mode  creating reads with a length of 101 or 151 bp. Sequencing chemistry v2 Illumina was used. Overall design: Examination of gene expressive levels  m6A enrichment levels  m6A single uncleotide sites and ythdf2 binding targets in normal and mettl3 or ythdf2 morphants zebrafish cells,,pubmed:28869969,,control zebrafish embryos m6A IP rep1,GSM2386177,,source name:zebrafish embryos|genotype/variation:normal|cell type:embryos cells|age:28hpf|tissue:trunk|enrichment antibody vendor:Synaptic Systems,control zebrafish embryos m6A IP rep1,Library strategy: MeRIP seq Reads were aligned to the zv9 genome assembly using TopHat v2.0.9 Genome build: zv9 Supplementary files format and content: m6A peak locus.xls: m6A peaks indentified by macs2 software Supplementary files format and content: reads count HTSeq.xls: Reads count for each gene of all samples,zebrafish embryos,,For MeRIP seq  mRNA was isolated from the trunck region of zebrafish embryos at 28hpf. For IP samples  mRNA was sonicated and subjected to IP using m6A antibody Synaptic Systems  202003. The RNA integrity was checked using an Agilent Bioanalyzer 2100. Approximately 2.5 µg of total RNA was then used for library preparation using a TruSeq™ RNA Sample Prep Kit v2 Illumina  San Diego  CA  USA according to the manufacturer’s protocol.The libraries were sequenced using HiSeq3000 Illumina in paired read mode  creating reads with a length of 151 bp.,,genotype/variation:normal|cell type:embryos cells|age:28hpf|tissue:trunk|enrichment antibody vendor:Synaptic Systems,GSM2386177,GSM2386177: control zebrafish embryos m6A IP rep1; Danio rerio; OTHER,GSM2386177,,1,For MeRIP seq  mRNA was isolated from the trunck region of zebrafish embryos at 28hpf. For IP samples  mRNA was sonicated and subjected to IP using m6A antibody Synaptic Systems  202003. The RNA integrity was checked using an Agilent Bioanalyzer 2100. Approximately 2.5 µg of total RNA was then used for library preparation using a TruSeq™ RNA Sample Prep Kit v2 Illumina  San Diego  CA  USA according to the manufacturer’s protocol.The libraries were sequenced using HiSeq3000 Illumina in paired read mode  creating reads with a length of 151 bp.,GEO Accession:GSM2386177,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,Illumina HiSeq 3000,,SRP092907,,,control_IP_rep1_1.fastq.gz control_IP_rep1_2.fastq.gz,fastq fastq,16582066208.0,54907504.0,GSM2386177 r1,0:151 1:151,A:4529890968;C:3812739433;G:4031512714;T:4204817283;N:3105810,151,151,,,4529890968,3812739433,4031512714,4204817283,3105810,SRX2336781,SRS1790179,SRA491654,GEO,Beijing Institute of Genomics (BIG) of Chinese Academy of Sciences (CAS),2,0.93701,0.95377,0.07009,0.07008,0.70972,0.71173,0.54711,0.54995,151,151,B,B,biological fallback assumption,illumina,hiseq_era,unknown,other,trueseq,bulk,bulk,bulk,,China,2016-11-08,Pharyngula,Embryo,Trunk,Surface Structure
68382,SRR17720609,SRX13883476,SRS11752245,SRP356476,PRJNA800053,Mapping the m1A  m5C  m6A and m7G Methylation Atlas in Zebrafish Brains under Hypoxic Conditions by MeRIP seq,GSE194284,Other,The epigenetic modifications play important regulatory roles in tissue development  maintenance of physiological functions and pathological process. RNA methylations  including newly identified m1A  m5C  m6A and m7G  are important epigenetic modifications. However  how these modifications are distributed in the transcriptome of vertebrate brains and whether their abundance is altered under pathological conditions are still poorly understood. In this study  we chose the model animal of zebrafish to conduct a systematic study to investigate the mRNA methylation atlas in the brain. By performing unbiased analyses of the m1A  m5C  m6A and m7G methylation of mRNA  we found that within the whole brain transcriptome  with the increase of the gene expression levels  the overall level of each of these four modifications on the related genes was also progressively increased. Further bioinformatics analysis indicated that the zebrafish brain has an abundance of m1A modifications. In the hypoxia treated zebrafish brains  the proportion of m1A is decreased  affecting the RNA splicing and zebrafish endogenous retroviruses. Our study presents the first comprehensive atlas of m1A  m5C  m6A and m7G in the epitranscriptome of the zebrafish brain and reveals the distribution of these modifications in mRNA under hypoxic conditions. These data provide an invaluable resource for further research on the involvement of m1A  m5C  m6A and m7G in the regulation of miRNA and repeat elements in vertebrates  and provide new thoughts to study the brain hypoxic injury on the aspect of epitranscriptome. Overall design: For each normoxia control and hypoxia experimental group  10 adult male zebrafish 3 mpf 4 mpf were chosen for the analyses. Three repeats of each experiment were performed and totally 30 fishes per group were collected. The brain tissues around 0.02 g per brain were then stored for further analysis in liquid nitrogen. For each analysis m1A  m5C  m6A  m7G and RNA Seq  the normoxia group and hypoxic group each consisted of 30 mixed brain tissues were analyzed parallelly by RNA seq and MeRIP Seq.,,pubmed:35135476,,m7G Normoxia IP,GSM5832287,,source name:zebrafish brain tissue|tissue:brain,m7G Normoxia IP,Paired end reads were harvested from an Illumina NovaSeq 6000 sequencer Cutadapt v1.9.3  command line software  was used to identify and trim 3’ adapter and low quality bases for rawdata. The clean data were mapped to the reference genome GRCz11 using Hisat2 software v2.0.4. For RNA seq: Guiding by the Ensembl GTF gene annotation file  Cuffdiff software part of cufflinks was used to obtain the gene level FPKM as the expression profiles of mRNA  fold change and p value were calculated based on FPKM For MeRIP seq: To identify the methylated sites on RNAs peaks  MACS software was utilized. Differentially methylated sites were identified by diffReps. The motif predictions of MeRIP Seq data were performed by utilizing a Perl script  findMotifGenome.pl  from HOMER software. Genome build: GRCz11 Supplementary files format and content: Gene expression valuesFPKM and Counts and methylated sites,zebrafish brain tissue,Adult wild type zebrafish Danio rerio were bred according to standard methods. For hypoxia treatment  we followed settings established in a previously published protocolYu et al 2011 with modified conditions. Briefly  zebrafish were transferred into a 1 L chamber containing 800 mL predeoxidated water O2: 0.3 0.4 mg/L. Oxygen in the water was then exhausted through the application of 8 L/min nitrogen until the dissolved oxygen value in the water was approximately 0.4 0.6 mg/L. Nitrogen 1 L/min was used to maintain the dissolved oxygen level in the water. post approximately 5 minutes  when the fish became motionless  the zebrafish brain were dissected on ice post anesthetizing to obtain brain tissues. The brain tissues were stored for further analysis in liquid nitrogen.,Ribo Zero rRNA Removal Kits Illumina  San Diego  CA  USA For RNA seq : TruSeq Stranded Total RNA Library Prep Kit Illumina  San Diego  CA  USA For MeRIP seq: GenSeqTM RNA IP Kit GenSeq Inc.  China and NEBNext® Ultra II Directional RNA Library Prep Kit New England Biolabs  Inc.  USA RNA seq and MeRIP seq,,tissue:brain,GSM5832287,GSM5832287: m7G Normoxia IP; Danio rerio; OTHER,GSM5832287 r1,GSM5832287,1,Ribo Zero rRNA Removal Kits Illumina  San Diego  CA  USA For RNA seq : TruSeq Stranded Total RNA Library Prep Kit Illumina  San Diego  CA  USA For MeRIP seq: GenSeqTM RNA IP Kit GenSeq Inc.  China and NEBNext® Ultra II Directional RNA Library Prep Kit New England Biolabs  Inc.  USA RNA seq and MeRIP seq,,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP356476,,loader:fastq load.py,m7G_Normoxia_IP_R1.fastq.gz m7G_Normoxia_IP_R2.fastq.gz,fastq fastq,6634104000.0,22113680.0,GSM5832287 r1,0:150 1:150,A:1396464650;C:1688319426;G:2250091870;T:1299109891;N:118163,150,150,,,1396464650,1688319426,2250091870,1299109891,118163,SRX13883476,SRS11752245,SRA1361294,Affiliated Hospital of Guangdong Medical University,Affiliated Hospital of Guangdong Medical University,2,0.9266,0.92801,0.07275,0.06866,0.89964,0.90601,0.65995,0.75535,150,150,B,B,biological fallback assumption,illumina,novaseq_era,full_length,rrna_depletion,nebnext,bulk,bulk,bulk,,China,2022-01-24,Adult,Adult,Brain,Nervous System
68383,SRR17720610,SRX13883475,SRS11752244,SRP356476,PRJNA800053,Mapping the m1A  m5C  m6A and m7G Methylation Atlas in Zebrafish Brains under Hypoxic Conditions by MeRIP seq,GSE194284,Other,The epigenetic modifications play important regulatory roles in tissue development  maintenance of physiological functions and pathological process. RNA methylations  including newly identified m1A  m5C  m6A and m7G  are important epigenetic modifications. However  how these modifications are distributed in the transcriptome of vertebrate brains and whether their abundance is altered under pathological conditions are still poorly understood. In this study  we chose the model animal of zebrafish to conduct a systematic study to investigate the mRNA methylation atlas in the brain. By performing unbiased analyses of the m1A  m5C  m6A and m7G methylation of mRNA  we found that within the whole brain transcriptome  with the increase of the gene expression levels  the overall level of each of these four modifications on the related genes was also progressively increased. Further bioinformatics analysis indicated that the zebrafish brain has an abundance of m1A modifications. In the hypoxia treated zebrafish brains  the proportion of m1A is decreased  affecting the RNA splicing and zebrafish endogenous retroviruses. Our study presents the first comprehensive atlas of m1A  m5C  m6A and m7G in the epitranscriptome of the zebrafish brain and reveals the distribution of these modifications in mRNA under hypoxic conditions. These data provide an invaluable resource for further research on the involvement of m1A  m5C  m6A and m7G in the regulation of miRNA and repeat elements in vertebrates  and provide new thoughts to study the brain hypoxic injury on the aspect of epitranscriptome. Overall design: For each normoxia control and hypoxia experimental group  10 adult male zebrafish 3 mpf 4 mpf were chosen for the analyses. Three repeats of each experiment were performed and totally 30 fishes per group were collected. The brain tissues around 0.02 g per brain were then stored for further analysis in liquid nitrogen. For each analysis m1A  m5C  m6A  m7G and RNA Seq  the normoxia group and hypoxic group each consisted of 30 mixed brain tissues were analyzed parallelly by RNA seq and MeRIP Seq.,,pubmed:35135476,,m7G Normoxia Input,GSM5832286,,source name:zebrafish brain tissue|tissue:brain,m7G Normoxia Input,Paired end reads were harvested from an Illumina NovaSeq 6000 sequencer Cutadapt v1.9.3  command line software  was used to identify and trim 3’ adapter and low quality bases for rawdata. The clean data were mapped to the reference genome GRCz11 using Hisat2 software v2.0.4. For RNA seq: Guiding by the Ensembl GTF gene annotation file  Cuffdiff software part of cufflinks was used to obtain the gene level FPKM as the expression profiles of mRNA  fold change and p value were calculated based on FPKM For MeRIP seq: To identify the methylated sites on RNAs peaks  MACS software was utilized. Differentially methylated sites were identified by diffReps. The motif predictions of MeRIP Seq data were performed by utilizing a Perl script  findMotifGenome.pl  from HOMER software. Genome build: GRCz11 Supplementary files format and content: Gene expression valuesFPKM and Counts and methylated sites,zebrafish brain tissue,Adult wild type zebrafish Danio rerio were bred according to standard methods. For hypoxia treatment  we followed settings established in a previously published protocolYu et al 2011 with modified conditions. Briefly  zebrafish were transferred into a 1 L chamber containing 800 mL predeoxidated water O2: 0.3 0.4 mg/L. Oxygen in the water was then exhausted through the application of 8 L/min nitrogen until the dissolved oxygen value in the water was approximately 0.4 0.6 mg/L. Nitrogen 1 L/min was used to maintain the dissolved oxygen level in the water. post approximately 5 minutes  when the fish became motionless  the zebrafish brain were dissected on ice post anesthetizing to obtain brain tissues. The brain tissues were stored for further analysis in liquid nitrogen.,Ribo Zero rRNA Removal Kits Illumina  San Diego  CA  USA For RNA seq : TruSeq Stranded Total RNA Library Prep Kit Illumina  San Diego  CA  USA For MeRIP seq: GenSeqTM RNA IP Kit GenSeq Inc.  China and NEBNext® Ultra II Directional RNA Library Prep Kit New England Biolabs  Inc.  USA RNA seq and MeRIP seq,,tissue:brain,GSM5832286,GSM5832286: m7G Normoxia Input; Danio rerio; OTHER,GSM5832286 r1,GSM5832286,1,Ribo Zero rRNA Removal Kits Illumina  San Diego  CA  USA For RNA seq : TruSeq Stranded Total RNA Library Prep Kit Illumina  San Diego  CA  USA For MeRIP seq: GenSeqTM RNA IP Kit GenSeq Inc.  China and NEBNext® Ultra II Directional RNA Library Prep Kit New England Biolabs  Inc.  USA RNA seq and MeRIP seq,,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP356476,,loader:fastq load.py,m7G_Normoxia_Input_R1.fastq.gz m7G_Normoxia_Input_R2.fastq.gz,fastq fastq,6839977800.0,22799926.0,GSM5832286 r1,0:150 1:150,A:1986780958;C:1411210049;G:1509902094;T:1932043482;N:41217,150,150,,,1986780958,1411210049,1509902094,1932043482,41217,SRX13883475,SRS11752244,SRA1361294,Affiliated Hospital of Guangdong Medical University,Affiliated Hospital of Guangdong Medical University,2,0.89709,0.89888,0.21747,0.21725,0.70021,0.69936,0.50469,0.51865,150,150,B,B,biological fallback assumption,illumina,novaseq_era,full_length,rrna_depletion,nebnext,bulk,bulk,bulk,,China,2022-01-24,Adult,Adult,Brain,Nervous System
68384,SRR17720611,SRX13883474,SRS11752243,SRP356476,PRJNA800053,Mapping the m1A  m5C  m6A and m7G Methylation Atlas in Zebrafish Brains under Hypoxic Conditions by MeRIP seq,GSE194284,Other,The epigenetic modifications play important regulatory roles in tissue development  maintenance of physiological functions and pathological process. RNA methylations  including newly identified m1A  m5C  m6A and m7G  are important epigenetic modifications. However  how these modifications are distributed in the transcriptome of vertebrate brains and whether their abundance is altered under pathological conditions are still poorly understood. In this study  we chose the model animal of zebrafish to conduct a systematic study to investigate the mRNA methylation atlas in the brain. By performing unbiased analyses of the m1A  m5C  m6A and m7G methylation of mRNA  we found that within the whole brain transcriptome  with the increase of the gene expression levels  the overall level of each of these four modifications on the related genes was also progressively increased. Further bioinformatics analysis indicated that the zebrafish brain has an abundance of m1A modifications. In the hypoxia treated zebrafish brains  the proportion of m1A is decreased  affecting the RNA splicing and zebrafish endogenous retroviruses. Our study presents the first comprehensive atlas of m1A  m5C  m6A and m7G in the epitranscriptome of the zebrafish brain and reveals the distribution of these modifications in mRNA under hypoxic conditions. These data provide an invaluable resource for further research on the involvement of m1A  m5C  m6A and m7G in the regulation of miRNA and repeat elements in vertebrates  and provide new thoughts to study the brain hypoxic injury on the aspect of epitranscriptome. Overall design: For each normoxia control and hypoxia experimental group  10 adult male zebrafish 3 mpf 4 mpf were chosen for the analyses. Three repeats of each experiment were performed and totally 30 fishes per group were collected. The brain tissues around 0.02 g per brain were then stored for further analysis in liquid nitrogen. For each analysis m1A  m5C  m6A  m7G and RNA Seq  the normoxia group and hypoxic group each consisted of 30 mixed brain tissues were analyzed parallelly by RNA seq and MeRIP Seq.,,pubmed:35135476,,m7G Hypoxia IP,GSM5832285,,source name:zebrafish brain tissue|tissue:brain,m7G Hypoxia IP,Paired end reads were harvested from an Illumina NovaSeq 6000 sequencer Cutadapt v1.9.3  command line software  was used to identify and trim 3’ adapter and low quality bases for rawdata. The clean data were mapped to the reference genome GRCz11 using Hisat2 software v2.0.4. For RNA seq: Guiding by the Ensembl GTF gene annotation file  Cuffdiff software part of cufflinks was used to obtain the gene level FPKM as the expression profiles of mRNA  fold change and p value were calculated based on FPKM For MeRIP seq: To identify the methylated sites on RNAs peaks  MACS software was utilized. Differentially methylated sites were identified by diffReps. The motif predictions of MeRIP Seq data were performed by utilizing a Perl script  findMotifGenome.pl  from HOMER software. Genome build: GRCz11 Supplementary files format and content: Gene expression valuesFPKM and Counts and methylated sites,zebrafish brain tissue,Adult wild type zebrafish Danio rerio were bred according to standard methods. For hypoxia treatment  we followed settings established in a previously published protocolYu et al 2011 with modified conditions. Briefly  zebrafish were transferred into a 1 L chamber containing 800 mL predeoxidated water O2: 0.3 0.4 mg/L. Oxygen in the water was then exhausted through the application of 8 L/min nitrogen until the dissolved oxygen value in the water was approximately 0.4 0.6 mg/L. Nitrogen 1 L/min was used to maintain the dissolved oxygen level in the water. post approximately 5 minutes  when the fish became motionless  the zebrafish brain were dissected on ice post anesthetizing to obtain brain tissues. The brain tissues were stored for further analysis in liquid nitrogen.,Ribo Zero rRNA Removal Kits Illumina  San Diego  CA  USA For RNA seq : TruSeq Stranded Total RNA Library Prep Kit Illumina  San Diego  CA  USA For MeRIP seq: GenSeqTM RNA IP Kit GenSeq Inc.  China and NEBNext® Ultra II Directional RNA Library Prep Kit New England Biolabs  Inc.  USA RNA seq and MeRIP seq,,tissue:brain,GSM5832285,GSM5832285: m7G Hypoxia IP; Danio rerio; OTHER,GSM5832285 r1,GSM5832285,1,Ribo Zero rRNA Removal Kits Illumina  San Diego  CA  USA For RNA seq : TruSeq Stranded Total RNA Library Prep Kit Illumina  San Diego  CA  USA For MeRIP seq: GenSeqTM RNA IP Kit GenSeq Inc.  China and NEBNext® Ultra II Directional RNA Library Prep Kit New England Biolabs  Inc.  USA RNA seq and MeRIP seq,,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP356476,,loader:fastq load.py,m7G_Hypoxia_IP_R1.fastq.gz m7G_Hypoxia_IP_R2.fastq.gz,fastq fastq,7282575000.0,24275250.0,GSM5832285 r1,0:150 1:150,A:1512757767;C:1913587260;G:2472336371;T:1383764924;N:128678,150,150,,,1512757767,1913587260,2472336371,1383764924,128678,SRX13883474,SRS11752243,SRA1361294,Affiliated Hospital of Guangdong Medical University,Affiliated Hospital of Guangdong Medical University,2,0.94526,0.9467,0.05248,0.05071,0.91946,0.91981,0.69303,0.78448,150,150,B,B,biological fallback assumption,illumina,novaseq_era,full_length,rrna_depletion,nebnext,bulk,bulk,bulk,,China,2022-01-24,Adult,Adult,Brain,Nervous System
68385,SRR17720612,SRX13883473,SRS11752241,SRP356476,PRJNA800053,Mapping the m1A  m5C  m6A and m7G Methylation Atlas in Zebrafish Brains under Hypoxic Conditions by MeRIP seq,GSE194284,Other,The epigenetic modifications play important regulatory roles in tissue development  maintenance of physiological functions and pathological process. RNA methylations  including newly identified m1A  m5C  m6A and m7G  are important epigenetic modifications. However  how these modifications are distributed in the transcriptome of vertebrate brains and whether their abundance is altered under pathological conditions are still poorly understood. In this study  we chose the model animal of zebrafish to conduct a systematic study to investigate the mRNA methylation atlas in the brain. By performing unbiased analyses of the m1A  m5C  m6A and m7G methylation of mRNA  we found that within the whole brain transcriptome  with the increase of the gene expression levels  the overall level of each of these four modifications on the related genes was also progressively increased. Further bioinformatics analysis indicated that the zebrafish brain has an abundance of m1A modifications. In the hypoxia treated zebrafish brains  the proportion of m1A is decreased  affecting the RNA splicing and zebrafish endogenous retroviruses. Our study presents the first comprehensive atlas of m1A  m5C  m6A and m7G in the epitranscriptome of the zebrafish brain and reveals the distribution of these modifications in mRNA under hypoxic conditions. These data provide an invaluable resource for further research on the involvement of m1A  m5C  m6A and m7G in the regulation of miRNA and repeat elements in vertebrates  and provide new thoughts to study the brain hypoxic injury on the aspect of epitranscriptome. Overall design: For each normoxia control and hypoxia experimental group  10 adult male zebrafish 3 mpf 4 mpf were chosen for the analyses. Three repeats of each experiment were performed and totally 30 fishes per group were collected. The brain tissues around 0.02 g per brain were then stored for further analysis in liquid nitrogen. For each analysis m1A  m5C  m6A  m7G and RNA Seq  the normoxia group and hypoxic group each consisted of 30 mixed brain tissues were analyzed parallelly by RNA seq and MeRIP Seq.,,pubmed:35135476,,m7G Hypoxia Input,GSM5832284,,source name:zebrafish brain tissue|tissue:brain,m7G Hypoxia Input,Paired end reads were harvested from an Illumina NovaSeq 6000 sequencer Cutadapt v1.9.3  command line software  was used to identify and trim 3’ adapter and low quality bases for rawdata. The clean data were mapped to the reference genome GRCz11 using Hisat2 software v2.0.4. For RNA seq: Guiding by the Ensembl GTF gene annotation file  Cuffdiff software part of cufflinks was used to obtain the gene level FPKM as the expression profiles of mRNA  fold change and p value were calculated based on FPKM For MeRIP seq: To identify the methylated sites on RNAs peaks  MACS software was utilized. Differentially methylated sites were identified by diffReps. The motif predictions of MeRIP Seq data were performed by utilizing a Perl script  findMotifGenome.pl  from HOMER software. Genome build: GRCz11 Supplementary files format and content: Gene expression valuesFPKM and Counts and methylated sites,zebrafish brain tissue,Adult wild type zebrafish Danio rerio were bred according to standard methods. For hypoxia treatment  we followed settings established in a previously published protocolYu et al 2011 with modified conditions. Briefly  zebrafish were transferred into a 1 L chamber containing 800 mL predeoxidated water O2: 0.3 0.4 mg/L. Oxygen in the water was then exhausted through the application of 8 L/min nitrogen until the dissolved oxygen value in the water was approximately 0.4 0.6 mg/L. Nitrogen 1 L/min was used to maintain the dissolved oxygen level in the water. post approximately 5 minutes  when the fish became motionless  the zebrafish brain were dissected on ice post anesthetizing to obtain brain tissues. The brain tissues were stored for further analysis in liquid nitrogen.,Ribo Zero rRNA Removal Kits Illumina  San Diego  CA  USA For RNA seq : TruSeq Stranded Total RNA Library Prep Kit Illumina  San Diego  CA  USA For MeRIP seq: GenSeqTM RNA IP Kit GenSeq Inc.  China and NEBNext® Ultra II Directional RNA Library Prep Kit New England Biolabs  Inc.  USA RNA seq and MeRIP seq,,tissue:brain,GSM5832284,GSM5832284: m7G Hypoxia Input; Danio rerio; OTHER,GSM5832284 r1,GSM5832284,1,Ribo Zero rRNA Removal Kits Illumina  San Diego  CA  USA For RNA seq : TruSeq Stranded Total RNA Library Prep Kit Illumina  San Diego  CA  USA For MeRIP seq: GenSeqTM RNA IP Kit GenSeq Inc.  China and NEBNext® Ultra II Directional RNA Library Prep Kit New England Biolabs  Inc.  USA RNA seq and MeRIP seq,,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP356476,,loader:fastq load.py,m7G_Hypoxia_Input_R1.fastq.gz m7G_Hypoxia_Input_R2.fastq.gz,fastq fastq,7492311600.0,24974372.0,GSM5832284 r1,0:150 1:150,A:2208863421;C:1521950436;G:1608371217;T:2153082842;N:43684,150,150,,,2208863421,1521950436,1608371217,2153082842,43684,SRX13883473,SRS11752241,SRA1361294,Affiliated Hospital of Guangdong Medical University,Affiliated Hospital of Guangdong Medical University,2,0.89264,0.89479,0.23239,0.23163,0.70496,0.70311,0.51741,0.52135,150,150,B,B,biological fallback assumption,illumina,novaseq_era,full_length,rrna_depletion,nebnext,bulk,bulk,bulk,,China,2022-01-24,Adult,Adult,Brain,Nervous System
68386,SRR17720613,SRX13883472,SRS11752242,SRP356476,PRJNA800053,Mapping the m1A  m5C  m6A and m7G Methylation Atlas in Zebrafish Brains under Hypoxic Conditions by MeRIP seq,GSE194284,Other,The epigenetic modifications play important regulatory roles in tissue development  maintenance of physiological functions and pathological process. RNA methylations  including newly identified m1A  m5C  m6A and m7G  are important epigenetic modifications. However  how these modifications are distributed in the transcriptome of vertebrate brains and whether their abundance is altered under pathological conditions are still poorly understood. In this study  we chose the model animal of zebrafish to conduct a systematic study to investigate the mRNA methylation atlas in the brain. By performing unbiased analyses of the m1A  m5C  m6A and m7G methylation of mRNA  we found that within the whole brain transcriptome  with the increase of the gene expression levels  the overall level of each of these four modifications on the related genes was also progressively increased. Further bioinformatics analysis indicated that the zebrafish brain has an abundance of m1A modifications. In the hypoxia treated zebrafish brains  the proportion of m1A is decreased  affecting the RNA splicing and zebrafish endogenous retroviruses. Our study presents the first comprehensive atlas of m1A  m5C  m6A and m7G in the epitranscriptome of the zebrafish brain and reveals the distribution of these modifications in mRNA under hypoxic conditions. These data provide an invaluable resource for further research on the involvement of m1A  m5C  m6A and m7G in the regulation of miRNA and repeat elements in vertebrates  and provide new thoughts to study the brain hypoxic injury on the aspect of epitranscriptome. Overall design: For each normoxia control and hypoxia experimental group  10 adult male zebrafish 3 mpf 4 mpf were chosen for the analyses. Three repeats of each experiment were performed and totally 30 fishes per group were collected. The brain tissues around 0.02 g per brain were then stored for further analysis in liquid nitrogen. For each analysis m1A  m5C  m6A  m7G and RNA Seq  the normoxia group and hypoxic group each consisted of 30 mixed brain tissues were analyzed parallelly by RNA seq and MeRIP Seq.,,pubmed:35135476,,m6A Normoxia IP,GSM5832283,,source name:zebrafish brain tissue|tissue:brain,m6A Normoxia IP,Paired end reads were harvested from an Illumina NovaSeq 6000 sequencer Cutadapt v1.9.3  command line software  was used to identify and trim 3’ adapter and low quality bases for rawdata. The clean data were mapped to the reference genome GRCz11 using Hisat2 software v2.0.4. For RNA seq: Guiding by the Ensembl GTF gene annotation file  Cuffdiff software part of cufflinks was used to obtain the gene level FPKM as the expression profiles of mRNA  fold change and p value were calculated based on FPKM For MeRIP seq: To identify the methylated sites on RNAs peaks  MACS software was utilized. Differentially methylated sites were identified by diffReps. The motif predictions of MeRIP Seq data were performed by utilizing a Perl script  findMotifGenome.pl  from HOMER software. Genome build: GRCz11 Supplementary files format and content: Gene expression valuesFPKM and Counts and methylated sites,zebrafish brain tissue,Adult wild type zebrafish Danio rerio were bred according to standard methods. For hypoxia treatment  we followed settings established in a previously published protocolYu et al 2011 with modified conditions. Briefly  zebrafish were transferred into a 1 L chamber containing 800 mL predeoxidated water O2: 0.3 0.4 mg/L. Oxygen in the water was then exhausted through the application of 8 L/min nitrogen until the dissolved oxygen value in the water was approximately 0.4 0.6 mg/L. Nitrogen 1 L/min was used to maintain the dissolved oxygen level in the water. post approximately 5 minutes  when the fish became motionless  the zebrafish brain were dissected on ice post anesthetizing to obtain brain tissues. The brain tissues were stored for further analysis in liquid nitrogen.,Ribo Zero rRNA Removal Kits Illumina  San Diego  CA  USA For RNA seq : TruSeq Stranded Total RNA Library Prep Kit Illumina  San Diego  CA  USA For MeRIP seq: GenSeqTM RNA IP Kit GenSeq Inc.  China and NEBNext® Ultra II Directional RNA Library Prep Kit New England Biolabs  Inc.  USA RNA seq and MeRIP seq,,tissue:brain,GSM5832283,GSM5832283: m6A Normoxia IP; Danio rerio; OTHER,GSM5832283 r1,GSM5832283,1,Ribo Zero rRNA Removal Kits Illumina  San Diego  CA  USA For RNA seq : TruSeq Stranded Total RNA Library Prep Kit Illumina  San Diego  CA  USA For MeRIP seq: GenSeqTM RNA IP Kit GenSeq Inc.  China and NEBNext® Ultra II Directional RNA Library Prep Kit New England Biolabs  Inc.  USA RNA seq and MeRIP seq,,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP356476,,,m6A_Normoxia_IP_R1.fastq.gz m6A_Normoxia_IP_R2.fastq.gz,fastq fastq,6583667700.0,21945559.0,GSM5832283 r1,0:150 1:150,A:1750230540;C:1500991424;G:1592129260;T:1740287575;N:28901,150,150,,,1750230540,1500991424,1592129260,1740287575,28901,SRX13883472,SRS11752242,SRA1361294,Affiliated Hospital of Guangdong Medical University,Affiliated Hospital of Guangdong Medical University,2,0.87682,0.87986,0.18203,0.17239,0.74671,0.7472,0.55638,0.55861,150,150,B,B,biological fallback assumption,illumina,novaseq_era,full_length,rrna_depletion,nebnext,bulk,bulk,bulk,,China,2022-01-24,Adult,Adult,Brain,Nervous System
68387,SRR17720614,SRX13883471,SRS11752240,SRP356476,PRJNA800053,Mapping the m1A  m5C  m6A and m7G Methylation Atlas in Zebrafish Brains under Hypoxic Conditions by MeRIP seq,GSE194284,Other,The epigenetic modifications play important regulatory roles in tissue development  maintenance of physiological functions and pathological process. RNA methylations  including newly identified m1A  m5C  m6A and m7G  are important epigenetic modifications. However  how these modifications are distributed in the transcriptome of vertebrate brains and whether their abundance is altered under pathological conditions are still poorly understood. In this study  we chose the model animal of zebrafish to conduct a systematic study to investigate the mRNA methylation atlas in the brain. By performing unbiased analyses of the m1A  m5C  m6A and m7G methylation of mRNA  we found that within the whole brain transcriptome  with the increase of the gene expression levels  the overall level of each of these four modifications on the related genes was also progressively increased. Further bioinformatics analysis indicated that the zebrafish brain has an abundance of m1A modifications. In the hypoxia treated zebrafish brains  the proportion of m1A is decreased  affecting the RNA splicing and zebrafish endogenous retroviruses. Our study presents the first comprehensive atlas of m1A  m5C  m6A and m7G in the epitranscriptome of the zebrafish brain and reveals the distribution of these modifications in mRNA under hypoxic conditions. These data provide an invaluable resource for further research on the involvement of m1A  m5C  m6A and m7G in the regulation of miRNA and repeat elements in vertebrates  and provide new thoughts to study the brain hypoxic injury on the aspect of epitranscriptome. Overall design: For each normoxia control and hypoxia experimental group  10 adult male zebrafish 3 mpf 4 mpf were chosen for the analyses. Three repeats of each experiment were performed and totally 30 fishes per group were collected. The brain tissues around 0.02 g per brain were then stored for further analysis in liquid nitrogen. For each analysis m1A  m5C  m6A  m7G and RNA Seq  the normoxia group and hypoxic group each consisted of 30 mixed brain tissues were analyzed parallelly by RNA seq and MeRIP Seq.,,pubmed:35135476,,m6A Normoxia Input,GSM5832282,,source name:zebrafish brain tissue|tissue:brain,m6A Normoxia Input,Paired end reads were harvested from an Illumina NovaSeq 6000 sequencer Cutadapt v1.9.3  command line software  was used to identify and trim 3’ adapter and low quality bases for rawdata. The clean data were mapped to the reference genome GRCz11 using Hisat2 software v2.0.4. For RNA seq: Guiding by the Ensembl GTF gene annotation file  Cuffdiff software part of cufflinks was used to obtain the gene level FPKM as the expression profiles of mRNA  fold change and p value were calculated based on FPKM For MeRIP seq: To identify the methylated sites on RNAs peaks  MACS software was utilized. Differentially methylated sites were identified by diffReps. The motif predictions of MeRIP Seq data were performed by utilizing a Perl script  findMotifGenome.pl  from HOMER software. Genome build: GRCz11 Supplementary files format and content: Gene expression valuesFPKM and Counts and methylated sites,zebrafish brain tissue,Adult wild type zebrafish Danio rerio were bred according to standard methods. For hypoxia treatment  we followed settings established in a previously published protocolYu et al 2011 with modified conditions. Briefly  zebrafish were transferred into a 1 L chamber containing 800 mL predeoxidated water O2: 0.3 0.4 mg/L. Oxygen in the water was then exhausted through the application of 8 L/min nitrogen until the dissolved oxygen value in the water was approximately 0.4 0.6 mg/L. Nitrogen 1 L/min was used to maintain the dissolved oxygen level in the water. post approximately 5 minutes  when the fish became motionless  the zebrafish brain were dissected on ice post anesthetizing to obtain brain tissues. The brain tissues were stored for further analysis in liquid nitrogen.,Ribo Zero rRNA Removal Kits Illumina  San Diego  CA  USA For RNA seq : TruSeq Stranded Total RNA Library Prep Kit Illumina  San Diego  CA  USA For MeRIP seq: GenSeqTM RNA IP Kit GenSeq Inc.  China and NEBNext® Ultra II Directional RNA Library Prep Kit New England Biolabs  Inc.  USA RNA seq and MeRIP seq,,tissue:brain,GSM5832282,GSM5832282: m6A Normoxia Input; Danio rerio; OTHER,GSM5832282 r1,GSM5832282,1,Ribo Zero rRNA Removal Kits Illumina  San Diego  CA  USA For RNA seq : TruSeq Stranded Total RNA Library Prep Kit Illumina  San Diego  CA  USA For MeRIP seq: GenSeqTM RNA IP Kit GenSeq Inc.  China and NEBNext® Ultra II Directional RNA Library Prep Kit New England Biolabs  Inc.  USA RNA seq and MeRIP seq,,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP356476,,loader:fastq load.py,m6A_Normoxia_Input_R1.fastq.gz m6A_Normoxia_Input_R2.fastq.gz,fastq fastq,6314076000.0,21046920.0,GSM5832282 r1,0:150 1:150,A:1815907910;C:1329572814;G:1378464668;T:1790102301;N:28307,150,150,,,1815907910,1329572814,1378464668,1790102301,28307,SRX13883471,SRS11752240,SRA1361294,Affiliated Hospital of Guangdong Medical University,Affiliated Hospital of Guangdong Medical University,2,0.91754,0.91835,0.21273,0.20993,0.69702,0.69625,0.53456,0.53763,150,150,B,B,biological fallback assumption,illumina,novaseq_era,full_length,rrna_depletion,nebnext,bulk,bulk,bulk,,China,2022-01-24,Adult,Adult,Brain,Nervous System
68388,SRR17720615,SRX13883470,SRS11752239,SRP356476,PRJNA800053,Mapping the m1A  m5C  m6A and m7G Methylation Atlas in Zebrafish Brains under Hypoxic Conditions by MeRIP seq,GSE194284,Other,The epigenetic modifications play important regulatory roles in tissue development  maintenance of physiological functions and pathological process. RNA methylations  including newly identified m1A  m5C  m6A and m7G  are important epigenetic modifications. However  how these modifications are distributed in the transcriptome of vertebrate brains and whether their abundance is altered under pathological conditions are still poorly understood. In this study  we chose the model animal of zebrafish to conduct a systematic study to investigate the mRNA methylation atlas in the brain. By performing unbiased analyses of the m1A  m5C  m6A and m7G methylation of mRNA  we found that within the whole brain transcriptome  with the increase of the gene expression levels  the overall level of each of these four modifications on the related genes was also progressively increased. Further bioinformatics analysis indicated that the zebrafish brain has an abundance of m1A modifications. In the hypoxia treated zebrafish brains  the proportion of m1A is decreased  affecting the RNA splicing and zebrafish endogenous retroviruses. Our study presents the first comprehensive atlas of m1A  m5C  m6A and m7G in the epitranscriptome of the zebrafish brain and reveals the distribution of these modifications in mRNA under hypoxic conditions. These data provide an invaluable resource for further research on the involvement of m1A  m5C  m6A and m7G in the regulation of miRNA and repeat elements in vertebrates  and provide new thoughts to study the brain hypoxic injury on the aspect of epitranscriptome. Overall design: For each normoxia control and hypoxia experimental group  10 adult male zebrafish 3 mpf 4 mpf were chosen for the analyses. Three repeats of each experiment were performed and totally 30 fishes per group were collected. The brain tissues around 0.02 g per brain were then stored for further analysis in liquid nitrogen. For each analysis m1A  m5C  m6A  m7G and RNA Seq  the normoxia group and hypoxic group each consisted of 30 mixed brain tissues were analyzed parallelly by RNA seq and MeRIP Seq.,,pubmed:35135476,,m6A Hypoxia IP,GSM5832281,,source name:zebrafish brain tissue|tissue:brain,m6A Hypoxia IP,Paired end reads were harvested from an Illumina NovaSeq 6000 sequencer Cutadapt v1.9.3  command line software  was used to identify and trim 3’ adapter and low quality bases for rawdata. The clean data were mapped to the reference genome GRCz11 using Hisat2 software v2.0.4. For RNA seq: Guiding by the Ensembl GTF gene annotation file  Cuffdiff software part of cufflinks was used to obtain the gene level FPKM as the expression profiles of mRNA  fold change and p value were calculated based on FPKM For MeRIP seq: To identify the methylated sites on RNAs peaks  MACS software was utilized. Differentially methylated sites were identified by diffReps. The motif predictions of MeRIP Seq data were performed by utilizing a Perl script  findMotifGenome.pl  from HOMER software. Genome build: GRCz11 Supplementary files format and content: Gene expression valuesFPKM and Counts and methylated sites,zebrafish brain tissue,Adult wild type zebrafish Danio rerio were bred according to standard methods. For hypoxia treatment  we followed settings established in a previously published protocolYu et al 2011 with modified conditions. Briefly  zebrafish were transferred into a 1 L chamber containing 800 mL predeoxidated water O2: 0.3 0.4 mg/L. Oxygen in the water was then exhausted through the application of 8 L/min nitrogen until the dissolved oxygen value in the water was approximately 0.4 0.6 mg/L. Nitrogen 1 L/min was used to maintain the dissolved oxygen level in the water. post approximately 5 minutes  when the fish became motionless  the zebrafish brain were dissected on ice post anesthetizing to obtain brain tissues. The brain tissues were stored for further analysis in liquid nitrogen.,Ribo Zero rRNA Removal Kits Illumina  San Diego  CA  USA For RNA seq : TruSeq Stranded Total RNA Library Prep Kit Illumina  San Diego  CA  USA For MeRIP seq: GenSeqTM RNA IP Kit GenSeq Inc.  China and NEBNext® Ultra II Directional RNA Library Prep Kit New England Biolabs  Inc.  USA RNA seq and MeRIP seq,,tissue:brain,GSM5832281,GSM5832281: m6A Hypoxia IP; Danio rerio; OTHER,GSM5832281 r1,GSM5832281,1,Ribo Zero rRNA Removal Kits Illumina  San Diego  CA  USA For RNA seq : TruSeq Stranded Total RNA Library Prep Kit Illumina  San Diego  CA  USA For MeRIP seq: GenSeqTM RNA IP Kit GenSeq Inc.  China and NEBNext® Ultra II Directional RNA Library Prep Kit New England Biolabs  Inc.  USA RNA seq and MeRIP seq,,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP356476,,,m6A_Hypoxia_IP_R1.fastq.gz m6A_Hypoxia_IP_R2.fastq.gz,fastq fastq,6490349100.0,21634497.0,GSM5832281 r1,0:150 1:150,A:1714144102;C:1483635762;G:1606044832;T:1686496024;N:28380,150,150,,,1714144102,1483635762,1606044832,1686496024,28380,SRX13883470,SRS11752239,SRA1361294,Affiliated Hospital of Guangdong Medical University,Affiliated Hospital of Guangdong Medical University,2,0.87598,0.88161,0.16783,0.16105,0.74588,0.74568,0.55361,0.55307,150,150,B,B,biological fallback assumption,illumina,novaseq_era,full_length,rrna_depletion,nebnext,bulk,bulk,bulk,,China,2022-01-24,Adult,Adult,Brain,Nervous System
68389,SRR17720616,SRX13883469,SRS11752237,SRP356476,PRJNA800053,Mapping the m1A  m5C  m6A and m7G Methylation Atlas in Zebrafish Brains under Hypoxic Conditions by MeRIP seq,GSE194284,Other,The epigenetic modifications play important regulatory roles in tissue development  maintenance of physiological functions and pathological process. RNA methylations  including newly identified m1A  m5C  m6A and m7G  are important epigenetic modifications. However  how these modifications are distributed in the transcriptome of vertebrate brains and whether their abundance is altered under pathological conditions are still poorly understood. In this study  we chose the model animal of zebrafish to conduct a systematic study to investigate the mRNA methylation atlas in the brain. By performing unbiased analyses of the m1A  m5C  m6A and m7G methylation of mRNA  we found that within the whole brain transcriptome  with the increase of the gene expression levels  the overall level of each of these four modifications on the related genes was also progressively increased. Further bioinformatics analysis indicated that the zebrafish brain has an abundance of m1A modifications. In the hypoxia treated zebrafish brains  the proportion of m1A is decreased  affecting the RNA splicing and zebrafish endogenous retroviruses. Our study presents the first comprehensive atlas of m1A  m5C  m6A and m7G in the epitranscriptome of the zebrafish brain and reveals the distribution of these modifications in mRNA under hypoxic conditions. These data provide an invaluable resource for further research on the involvement of m1A  m5C  m6A and m7G in the regulation of miRNA and repeat elements in vertebrates  and provide new thoughts to study the brain hypoxic injury on the aspect of epitranscriptome. Overall design: For each normoxia control and hypoxia experimental group  10 adult male zebrafish 3 mpf 4 mpf were chosen for the analyses. Three repeats of each experiment were performed and totally 30 fishes per group were collected. The brain tissues around 0.02 g per brain were then stored for further analysis in liquid nitrogen. For each analysis m1A  m5C  m6A  m7G and RNA Seq  the normoxia group and hypoxic group each consisted of 30 mixed brain tissues were analyzed parallelly by RNA seq and MeRIP Seq.,,pubmed:35135476,,m6A Hypoxia Input,GSM5832280,,source name:zebrafish brain tissue|tissue:brain,m6A Hypoxia Input,Paired end reads were harvested from an Illumina NovaSeq 6000 sequencer Cutadapt v1.9.3  command line software  was used to identify and trim 3’ adapter and low quality bases for rawdata. The clean data were mapped to the reference genome GRCz11 using Hisat2 software v2.0.4. For RNA seq: Guiding by the Ensembl GTF gene annotation file  Cuffdiff software part of cufflinks was used to obtain the gene level FPKM as the expression profiles of mRNA  fold change and p value were calculated based on FPKM For MeRIP seq: To identify the methylated sites on RNAs peaks  MACS software was utilized. Differentially methylated sites were identified by diffReps. The motif predictions of MeRIP Seq data were performed by utilizing a Perl script  findMotifGenome.pl  from HOMER software. Genome build: GRCz11 Supplementary files format and content: Gene expression valuesFPKM and Counts and methylated sites,zebrafish brain tissue,Adult wild type zebrafish Danio rerio were bred according to standard methods. For hypoxia treatment  we followed settings established in a previously published protocolYu et al 2011 with modified conditions. Briefly  zebrafish were transferred into a 1 L chamber containing 800 mL predeoxidated water O2: 0.3 0.4 mg/L. Oxygen in the water was then exhausted through the application of 8 L/min nitrogen until the dissolved oxygen value in the water was approximately 0.4 0.6 mg/L. Nitrogen 1 L/min was used to maintain the dissolved oxygen level in the water. post approximately 5 minutes  when the fish became motionless  the zebrafish brain were dissected on ice post anesthetizing to obtain brain tissues. The brain tissues were stored for further analysis in liquid nitrogen.,Ribo Zero rRNA Removal Kits Illumina  San Diego  CA  USA For RNA seq : TruSeq Stranded Total RNA Library Prep Kit Illumina  San Diego  CA  USA For MeRIP seq: GenSeqTM RNA IP Kit GenSeq Inc.  China and NEBNext® Ultra II Directional RNA Library Prep Kit New England Biolabs  Inc.  USA RNA seq and MeRIP seq,,tissue:brain,GSM5832280,GSM5832280: m6A Hypoxia Input; Danio rerio; OTHER,GSM5832280 r1,GSM5832280,1,Ribo Zero rRNA Removal Kits Illumina  San Diego  CA  USA For RNA seq : TruSeq Stranded Total RNA Library Prep Kit Illumina  San Diego  CA  USA For MeRIP seq: GenSeqTM RNA IP Kit GenSeq Inc.  China and NEBNext® Ultra II Directional RNA Library Prep Kit New England Biolabs  Inc.  USA RNA seq and MeRIP seq,,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP356476,,loader:fastq load.py,m6A_Hypoxia_Input_R1.fastq.gz m6A_Hypoxia_Input_R2.fastq.gz,fastq fastq,7054483800.0,23514946.0,GSM5832280 r1,0:150 1:150,A:2000164754;C:1513377207;G:1570441784;T:1970468107;N:31948,150,150,,,2000164754,1513377207,1570441784,1970468107,31948,SRX13883469,SRS11752237,SRA1361294,Affiliated Hospital of Guangdong Medical University,Affiliated Hospital of Guangdong Medical University,2,0.91916,0.91997,0.21403,0.21158,0.69716,0.69503,0.52616,0.51982,150,150,B,B,biological fallback assumption,illumina,novaseq_era,full_length,rrna_depletion,nebnext,bulk,bulk,bulk,,China,2022-01-24,Adult,Adult,Brain,Nervous System
68390,SRR17720617,SRX13883468,SRS11752238,SRP356476,PRJNA800053,Mapping the m1A  m5C  m6A and m7G Methylation Atlas in Zebrafish Brains under Hypoxic Conditions by MeRIP seq,GSE194284,Other,The epigenetic modifications play important regulatory roles in tissue development  maintenance of physiological functions and pathological process. RNA methylations  including newly identified m1A  m5C  m6A and m7G  are important epigenetic modifications. However  how these modifications are distributed in the transcriptome of vertebrate brains and whether their abundance is altered under pathological conditions are still poorly understood. In this study  we chose the model animal of zebrafish to conduct a systematic study to investigate the mRNA methylation atlas in the brain. By performing unbiased analyses of the m1A  m5C  m6A and m7G methylation of mRNA  we found that within the whole brain transcriptome  with the increase of the gene expression levels  the overall level of each of these four modifications on the related genes was also progressively increased. Further bioinformatics analysis indicated that the zebrafish brain has an abundance of m1A modifications. In the hypoxia treated zebrafish brains  the proportion of m1A is decreased  affecting the RNA splicing and zebrafish endogenous retroviruses. Our study presents the first comprehensive atlas of m1A  m5C  m6A and m7G in the epitranscriptome of the zebrafish brain and reveals the distribution of these modifications in mRNA under hypoxic conditions. These data provide an invaluable resource for further research on the involvement of m1A  m5C  m6A and m7G in the regulation of miRNA and repeat elements in vertebrates  and provide new thoughts to study the brain hypoxic injury on the aspect of epitranscriptome. Overall design: For each normoxia control and hypoxia experimental group  10 adult male zebrafish 3 mpf 4 mpf were chosen for the analyses. Three repeats of each experiment were performed and totally 30 fishes per group were collected. The brain tissues around 0.02 g per brain were then stored for further analysis in liquid nitrogen. For each analysis m1A  m5C  m6A  m7G and RNA Seq  the normoxia group and hypoxic group each consisted of 30 mixed brain tissues were analyzed parallelly by RNA seq and MeRIP Seq.,,pubmed:35135476,,m5C Normoxia IP,GSM5832279,,source name:zebrafish brain tissue|tissue:brain,m5C Normoxia IP,Paired end reads were harvested from an Illumina NovaSeq 6000 sequencer Cutadapt v1.9.3  command line software  was used to identify and trim 3’ adapter and low quality bases for rawdata. The clean data were mapped to the reference genome GRCz11 using Hisat2 software v2.0.4. For RNA seq: Guiding by the Ensembl GTF gene annotation file  Cuffdiff software part of cufflinks was used to obtain the gene level FPKM as the expression profiles of mRNA  fold change and p value were calculated based on FPKM For MeRIP seq: To identify the methylated sites on RNAs peaks  MACS software was utilized. Differentially methylated sites were identified by diffReps. The motif predictions of MeRIP Seq data were performed by utilizing a Perl script  findMotifGenome.pl  from HOMER software. Genome build: GRCz11 Supplementary files format and content: Gene expression valuesFPKM and Counts and methylated sites,zebrafish brain tissue,Adult wild type zebrafish Danio rerio were bred according to standard methods. For hypoxia treatment  we followed settings established in a previously published protocolYu et al 2011 with modified conditions. Briefly  zebrafish were transferred into a 1 L chamber containing 800 mL predeoxidated water O2: 0.3 0.4 mg/L. Oxygen in the water was then exhausted through the application of 8 L/min nitrogen until the dissolved oxygen value in the water was approximately 0.4 0.6 mg/L. Nitrogen 1 L/min was used to maintain the dissolved oxygen level in the water. post approximately 5 minutes  when the fish became motionless  the zebrafish brain were dissected on ice post anesthetizing to obtain brain tissues. The brain tissues were stored for further analysis in liquid nitrogen.,Ribo Zero rRNA Removal Kits Illumina  San Diego  CA  USA For RNA seq : TruSeq Stranded Total RNA Library Prep Kit Illumina  San Diego  CA  USA For MeRIP seq: GenSeqTM RNA IP Kit GenSeq Inc.  China and NEBNext® Ultra II Directional RNA Library Prep Kit New England Biolabs  Inc.  USA RNA seq and MeRIP seq,,tissue:brain,GSM5832279,GSM5832279: m5C Normoxia IP; Danio rerio; OTHER,GSM5832279 r1,GSM5832279,1,Ribo Zero rRNA Removal Kits Illumina  San Diego  CA  USA For RNA seq : TruSeq Stranded Total RNA Library Prep Kit Illumina  San Diego  CA  USA For MeRIP seq: GenSeqTM RNA IP Kit GenSeq Inc.  China and NEBNext® Ultra II Directional RNA Library Prep Kit New England Biolabs  Inc.  USA RNA seq and MeRIP seq,,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP356476,,loader:fastq load.py,m5C_Normoxia_IP_R1.fastq.gz m5C_Normoxia_IP_R2.fastq.gz,fastq fastq,7385723100.0,24619077.0,GSM5832279 r1,0:150 1:150,A:1779838658;C:1853060139;G:2010257354;T:1742536078;N:30871,150,150,,,1779838658,1853060139,2010257354,1742536078,30871,SRX13883468,SRS11752238,SRA1361294,Affiliated Hospital of Guangdong Medical University,Affiliated Hospital of Guangdong Medical University,2,0.88617,0.87633,0.42619,0.41974,0.71792,0.74016,0.69827,0.68649,150,150,B,B,biological fallback assumption,illumina,novaseq_era,full_length,rrna_depletion,nebnext,bulk,bulk,bulk,,China,2022-01-24,Adult,Adult,Brain,Nervous System
68391,SRR17720618,SRX13883467,SRS11752236,SRP356476,PRJNA800053,Mapping the m1A  m5C  m6A and m7G Methylation Atlas in Zebrafish Brains under Hypoxic Conditions by MeRIP seq,GSE194284,Other,The epigenetic modifications play important regulatory roles in tissue development  maintenance of physiological functions and pathological process. RNA methylations  including newly identified m1A  m5C  m6A and m7G  are important epigenetic modifications. However  how these modifications are distributed in the transcriptome of vertebrate brains and whether their abundance is altered under pathological conditions are still poorly understood. In this study  we chose the model animal of zebrafish to conduct a systematic study to investigate the mRNA methylation atlas in the brain. By performing unbiased analyses of the m1A  m5C  m6A and m7G methylation of mRNA  we found that within the whole brain transcriptome  with the increase of the gene expression levels  the overall level of each of these four modifications on the related genes was also progressively increased. Further bioinformatics analysis indicated that the zebrafish brain has an abundance of m1A modifications. In the hypoxia treated zebrafish brains  the proportion of m1A is decreased  affecting the RNA splicing and zebrafish endogenous retroviruses. Our study presents the first comprehensive atlas of m1A  m5C  m6A and m7G in the epitranscriptome of the zebrafish brain and reveals the distribution of these modifications in mRNA under hypoxic conditions. These data provide an invaluable resource for further research on the involvement of m1A  m5C  m6A and m7G in the regulation of miRNA and repeat elements in vertebrates  and provide new thoughts to study the brain hypoxic injury on the aspect of epitranscriptome. Overall design: For each normoxia control and hypoxia experimental group  10 adult male zebrafish 3 mpf 4 mpf were chosen for the analyses. Three repeats of each experiment were performed and totally 30 fishes per group were collected. The brain tissues around 0.02 g per brain were then stored for further analysis in liquid nitrogen. For each analysis m1A  m5C  m6A  m7G and RNA Seq  the normoxia group and hypoxic group each consisted of 30 mixed brain tissues were analyzed parallelly by RNA seq and MeRIP Seq.,,pubmed:35135476,,m5C Normoxia Input,GSM5832278,,source name:zebrafish brain tissue|tissue:brain,m5C Normoxia Input,Paired end reads were harvested from an Illumina NovaSeq 6000 sequencer Cutadapt v1.9.3  command line software  was used to identify and trim 3’ adapter and low quality bases for rawdata. The clean data were mapped to the reference genome GRCz11 using Hisat2 software v2.0.4. For RNA seq: Guiding by the Ensembl GTF gene annotation file  Cuffdiff software part of cufflinks was used to obtain the gene level FPKM as the expression profiles of mRNA  fold change and p value were calculated based on FPKM For MeRIP seq: To identify the methylated sites on RNAs peaks  MACS software was utilized. Differentially methylated sites were identified by diffReps. The motif predictions of MeRIP Seq data were performed by utilizing a Perl script  findMotifGenome.pl  from HOMER software. Genome build: GRCz11 Supplementary files format and content: Gene expression valuesFPKM and Counts and methylated sites,zebrafish brain tissue,Adult wild type zebrafish Danio rerio were bred according to standard methods. For hypoxia treatment  we followed settings established in a previously published protocolYu et al 2011 with modified conditions. Briefly  zebrafish were transferred into a 1 L chamber containing 800 mL predeoxidated water O2: 0.3 0.4 mg/L. Oxygen in the water was then exhausted through the application of 8 L/min nitrogen until the dissolved oxygen value in the water was approximately 0.4 0.6 mg/L. Nitrogen 1 L/min was used to maintain the dissolved oxygen level in the water. post approximately 5 minutes  when the fish became motionless  the zebrafish brain were dissected on ice post anesthetizing to obtain brain tissues. The brain tissues were stored for further analysis in liquid nitrogen.,Ribo Zero rRNA Removal Kits Illumina  San Diego  CA  USA For RNA seq : TruSeq Stranded Total RNA Library Prep Kit Illumina  San Diego  CA  USA For MeRIP seq: GenSeqTM RNA IP Kit GenSeq Inc.  China and NEBNext® Ultra II Directional RNA Library Prep Kit New England Biolabs  Inc.  USA RNA seq and MeRIP seq,,tissue:brain,GSM5832278,GSM5832278: m5C Normoxia Input; Danio rerio; OTHER,GSM5832278 r1,GSM5832278,1,Ribo Zero rRNA Removal Kits Illumina  San Diego  CA  USA For RNA seq : TruSeq Stranded Total RNA Library Prep Kit Illumina  San Diego  CA  USA For MeRIP seq: GenSeqTM RNA IP Kit GenSeq Inc.  China and NEBNext® Ultra II Directional RNA Library Prep Kit New England Biolabs  Inc.  USA RNA seq and MeRIP seq,,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP356476,,loader:fastq load.py,m5C_Normoxia_Input_R1.fastq.gz m5C_Normoxia_Input_R2.fastq.gz,fastq fastq,6594811500.0,21982705.0,GSM5832278 r1,0:150 1:150,A:1941068575;C:1341378672;G:1414823713;T:1897501503;N:39037,150,150,,,1941068575,1341378672,1414823713,1897501503,39037,SRX13883467,SRS11752236,SRA1361294,Affiliated Hospital of Guangdong Medical University,Affiliated Hospital of Guangdong Medical University,2,0.89423,0.89602,0.22595,0.2243,0.70212,0.70112,0.52954,0.52253,150,150,B,B,biological fallback assumption,illumina,novaseq_era,full_length,rrna_depletion,nebnext,bulk,bulk,bulk,,China,2022-01-24,Adult,Adult,Brain,Nervous System
68392,SRR17720619,SRX13883466,SRS11752235,SRP356476,PRJNA800053,Mapping the m1A  m5C  m6A and m7G Methylation Atlas in Zebrafish Brains under Hypoxic Conditions by MeRIP seq,GSE194284,Other,The epigenetic modifications play important regulatory roles in tissue development  maintenance of physiological functions and pathological process. RNA methylations  including newly identified m1A  m5C  m6A and m7G  are important epigenetic modifications. However  how these modifications are distributed in the transcriptome of vertebrate brains and whether their abundance is altered under pathological conditions are still poorly understood. In this study  we chose the model animal of zebrafish to conduct a systematic study to investigate the mRNA methylation atlas in the brain. By performing unbiased analyses of the m1A  m5C  m6A and m7G methylation of mRNA  we found that within the whole brain transcriptome  with the increase of the gene expression levels  the overall level of each of these four modifications on the related genes was also progressively increased. Further bioinformatics analysis indicated that the zebrafish brain has an abundance of m1A modifications. In the hypoxia treated zebrafish brains  the proportion of m1A is decreased  affecting the RNA splicing and zebrafish endogenous retroviruses. Our study presents the first comprehensive atlas of m1A  m5C  m6A and m7G in the epitranscriptome of the zebrafish brain and reveals the distribution of these modifications in mRNA under hypoxic conditions. These data provide an invaluable resource for further research on the involvement of m1A  m5C  m6A and m7G in the regulation of miRNA and repeat elements in vertebrates  and provide new thoughts to study the brain hypoxic injury on the aspect of epitranscriptome. Overall design: For each normoxia control and hypoxia experimental group  10 adult male zebrafish 3 mpf 4 mpf were chosen for the analyses. Three repeats of each experiment were performed and totally 30 fishes per group were collected. The brain tissues around 0.02 g per brain were then stored for further analysis in liquid nitrogen. For each analysis m1A  m5C  m6A  m7G and RNA Seq  the normoxia group and hypoxic group each consisted of 30 mixed brain tissues were analyzed parallelly by RNA seq and MeRIP Seq.,,pubmed:35135476,,m5C Hypoxia IP,GSM5832277,,source name:zebrafish brain tissue|tissue:brain,m5C Hypoxia IP,Paired end reads were harvested from an Illumina NovaSeq 6000 sequencer Cutadapt v1.9.3  command line software  was used to identify and trim 3’ adapter and low quality bases for rawdata. The clean data were mapped to the reference genome GRCz11 using Hisat2 software v2.0.4. For RNA seq: Guiding by the Ensembl GTF gene annotation file  Cuffdiff software part of cufflinks was used to obtain the gene level FPKM as the expression profiles of mRNA  fold change and p value were calculated based on FPKM For MeRIP seq: To identify the methylated sites on RNAs peaks  MACS software was utilized. Differentially methylated sites were identified by diffReps. The motif predictions of MeRIP Seq data were performed by utilizing a Perl script  findMotifGenome.pl  from HOMER software. Genome build: GRCz11 Supplementary files format and content: Gene expression valuesFPKM and Counts and methylated sites,zebrafish brain tissue,Adult wild type zebrafish Danio rerio were bred according to standard methods. For hypoxia treatment  we followed settings established in a previously published protocolYu et al 2011 with modified conditions. Briefly  zebrafish were transferred into a 1 L chamber containing 800 mL predeoxidated water O2: 0.3 0.4 mg/L. Oxygen in the water was then exhausted through the application of 8 L/min nitrogen until the dissolved oxygen value in the water was approximately 0.4 0.6 mg/L. Nitrogen 1 L/min was used to maintain the dissolved oxygen level in the water. post approximately 5 minutes  when the fish became motionless  the zebrafish brain were dissected on ice post anesthetizing to obtain brain tissues. The brain tissues were stored for further analysis in liquid nitrogen.,Ribo Zero rRNA Removal Kits Illumina  San Diego  CA  USA For RNA seq : TruSeq Stranded Total RNA Library Prep Kit Illumina  San Diego  CA  USA For MeRIP seq: GenSeqTM RNA IP Kit GenSeq Inc.  China and NEBNext® Ultra II Directional RNA Library Prep Kit New England Biolabs  Inc.  USA RNA seq and MeRIP seq,,tissue:brain,GSM5832277,GSM5832277: m5C Hypoxia IP; Danio rerio; OTHER,GSM5832277 r1,GSM5832277,1,Ribo Zero rRNA Removal Kits Illumina  San Diego  CA  USA For RNA seq : TruSeq Stranded Total RNA Library Prep Kit Illumina  San Diego  CA  USA For MeRIP seq: GenSeqTM RNA IP Kit GenSeq Inc.  China and NEBNext® Ultra II Directional RNA Library Prep Kit New England Biolabs  Inc.  USA RNA seq and MeRIP seq,,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP356476,,loader:fastq load.py,m5C_Hypoxia_IP_R1.fastq.gz m5C_Hypoxia_IP_R2.fastq.gz,fastq fastq,7837067100.0,26123557.0,GSM5832277 r1,0:150 1:150,A:1881167242;C:1973697370;G:2143523979;T:1838645554;N:32955,150,150,,,1881167242,1973697370,2143523979,1838645554,32955,SRX13883466,SRS11752235,SRA1361294,Affiliated Hospital of Guangdong Medical University,Affiliated Hospital of Guangdong Medical University,2,0.87593,0.87108,0.45438,0.45093,0.71664,0.7321,0.67861,0.69742,150,150,B,B,biological fallback assumption,illumina,novaseq_era,full_length,rrna_depletion,nebnext,bulk,bulk,bulk,,China,2022-01-24,Adult,Adult,Brain,Nervous System
68393,SRR17720620,SRX13883465,SRS11752233,SRP356476,PRJNA800053,Mapping the m1A  m5C  m6A and m7G Methylation Atlas in Zebrafish Brains under Hypoxic Conditions by MeRIP seq,GSE194284,Other,The epigenetic modifications play important regulatory roles in tissue development  maintenance of physiological functions and pathological process. RNA methylations  including newly identified m1A  m5C  m6A and m7G  are important epigenetic modifications. However  how these modifications are distributed in the transcriptome of vertebrate brains and whether their abundance is altered under pathological conditions are still poorly understood. In this study  we chose the model animal of zebrafish to conduct a systematic study to investigate the mRNA methylation atlas in the brain. By performing unbiased analyses of the m1A  m5C  m6A and m7G methylation of mRNA  we found that within the whole brain transcriptome  with the increase of the gene expression levels  the overall level of each of these four modifications on the related genes was also progressively increased. Further bioinformatics analysis indicated that the zebrafish brain has an abundance of m1A modifications. In the hypoxia treated zebrafish brains  the proportion of m1A is decreased  affecting the RNA splicing and zebrafish endogenous retroviruses. Our study presents the first comprehensive atlas of m1A  m5C  m6A and m7G in the epitranscriptome of the zebrafish brain and reveals the distribution of these modifications in mRNA under hypoxic conditions. These data provide an invaluable resource for further research on the involvement of m1A  m5C  m6A and m7G in the regulation of miRNA and repeat elements in vertebrates  and provide new thoughts to study the brain hypoxic injury on the aspect of epitranscriptome. Overall design: For each normoxia control and hypoxia experimental group  10 adult male zebrafish 3 mpf 4 mpf were chosen for the analyses. Three repeats of each experiment were performed and totally 30 fishes per group were collected. The brain tissues around 0.02 g per brain were then stored for further analysis in liquid nitrogen. For each analysis m1A  m5C  m6A  m7G and RNA Seq  the normoxia group and hypoxic group each consisted of 30 mixed brain tissues were analyzed parallelly by RNA seq and MeRIP Seq.,,pubmed:35135476,,m5C Hypoxia Input,GSM5832276,,source name:zebrafish brain tissue|tissue:brain,m5C Hypoxia Input,Paired end reads were harvested from an Illumina NovaSeq 6000 sequencer Cutadapt v1.9.3  command line software  was used to identify and trim 3’ adapter and low quality bases for rawdata. The clean data were mapped to the reference genome GRCz11 using Hisat2 software v2.0.4. For RNA seq: Guiding by the Ensembl GTF gene annotation file  Cuffdiff software part of cufflinks was used to obtain the gene level FPKM as the expression profiles of mRNA  fold change and p value were calculated based on FPKM For MeRIP seq: To identify the methylated sites on RNAs peaks  MACS software was utilized. Differentially methylated sites were identified by diffReps. The motif predictions of MeRIP Seq data were performed by utilizing a Perl script  findMotifGenome.pl  from HOMER software. Genome build: GRCz11 Supplementary files format and content: Gene expression valuesFPKM and Counts and methylated sites,zebrafish brain tissue,Adult wild type zebrafish Danio rerio were bred according to standard methods. For hypoxia treatment  we followed settings established in a previously published protocolYu et al 2011 with modified conditions. Briefly  zebrafish were transferred into a 1 L chamber containing 800 mL predeoxidated water O2: 0.3 0.4 mg/L. Oxygen in the water was then exhausted through the application of 8 L/min nitrogen until the dissolved oxygen value in the water was approximately 0.4 0.6 mg/L. Nitrogen 1 L/min was used to maintain the dissolved oxygen level in the water. post approximately 5 minutes  when the fish became motionless  the zebrafish brain were dissected on ice post anesthetizing to obtain brain tissues. The brain tissues were stored for further analysis in liquid nitrogen.,Ribo Zero rRNA Removal Kits Illumina  San Diego  CA  USA For RNA seq : TruSeq Stranded Total RNA Library Prep Kit Illumina  San Diego  CA  USA For MeRIP seq: GenSeqTM RNA IP Kit GenSeq Inc.  China and NEBNext® Ultra II Directional RNA Library Prep Kit New England Biolabs  Inc.  USA RNA seq and MeRIP seq,,tissue:brain,GSM5832276,GSM5832276: m5C Hypoxia Input; Danio rerio; OTHER,GSM5832276 r1,GSM5832276,1,Ribo Zero rRNA Removal Kits Illumina  San Diego  CA  USA For RNA seq : TruSeq Stranded Total RNA Library Prep Kit Illumina  San Diego  CA  USA For MeRIP seq: GenSeqTM RNA IP Kit GenSeq Inc.  China and NEBNext® Ultra II Directional RNA Library Prep Kit New England Biolabs  Inc.  USA RNA seq and MeRIP seq,,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP356476,,loader:fastq load.py,m5C_Hypoxia_Input_R1.fastq.gz m5C_Hypoxia_Input_R2.fastq.gz,fastq fastq,8696183100.0,28987277.0,GSM5832276 r1,0:150 1:150,A:2564166903;C:1771446935;G:1858965930;T:2501551942;N:51390,150,150,,,2564166903,1771446935,1858965930,2501551942,51390,SRX13883465,SRS11752233,SRA1361294,Affiliated Hospital of Guangdong Medical University,Affiliated Hospital of Guangdong Medical University,2,0.89379,0.89585,0.23898,0.23828,0.70272,0.70278,0.52965,0.50047,150,150,B,B,biological fallback assumption,illumina,novaseq_era,full_length,rrna_depletion,nebnext,bulk,bulk,bulk,,China,2022-01-24,Adult,Adult,Brain,Nervous System
68394,SRR17720621,SRX13883464,SRS11752234,SRP356476,PRJNA800053,Mapping the m1A  m5C  m6A and m7G Methylation Atlas in Zebrafish Brains under Hypoxic Conditions by MeRIP seq,GSE194284,Other,The epigenetic modifications play important regulatory roles in tissue development  maintenance of physiological functions and pathological process. RNA methylations  including newly identified m1A  m5C  m6A and m7G  are important epigenetic modifications. However  how these modifications are distributed in the transcriptome of vertebrate brains and whether their abundance is altered under pathological conditions are still poorly understood. In this study  we chose the model animal of zebrafish to conduct a systematic study to investigate the mRNA methylation atlas in the brain. By performing unbiased analyses of the m1A  m5C  m6A and m7G methylation of mRNA  we found that within the whole brain transcriptome  with the increase of the gene expression levels  the overall level of each of these four modifications on the related genes was also progressively increased. Further bioinformatics analysis indicated that the zebrafish brain has an abundance of m1A modifications. In the hypoxia treated zebrafish brains  the proportion of m1A is decreased  affecting the RNA splicing and zebrafish endogenous retroviruses. Our study presents the first comprehensive atlas of m1A  m5C  m6A and m7G in the epitranscriptome of the zebrafish brain and reveals the distribution of these modifications in mRNA under hypoxic conditions. These data provide an invaluable resource for further research on the involvement of m1A  m5C  m6A and m7G in the regulation of miRNA and repeat elements in vertebrates  and provide new thoughts to study the brain hypoxic injury on the aspect of epitranscriptome. Overall design: For each normoxia control and hypoxia experimental group  10 adult male zebrafish 3 mpf 4 mpf were chosen for the analyses. Three repeats of each experiment were performed and totally 30 fishes per group were collected. The brain tissues around 0.02 g per brain were then stored for further analysis in liquid nitrogen. For each analysis m1A  m5C  m6A  m7G and RNA Seq  the normoxia group and hypoxic group each consisted of 30 mixed brain tissues were analyzed parallelly by RNA seq and MeRIP Seq.,,pubmed:35135476,,m1A Normoxia IP,GSM5832275,,source name:zebrafish brain tissue|tissue:brain,m1A Normoxia IP,Paired end reads were harvested from an Illumina NovaSeq 6000 sequencer Cutadapt v1.9.3  command line software  was used to identify and trim 3’ adapter and low quality bases for rawdata. The clean data were mapped to the reference genome GRCz11 using Hisat2 software v2.0.4. For RNA seq: Guiding by the Ensembl GTF gene annotation file  Cuffdiff software part of cufflinks was used to obtain the gene level FPKM as the expression profiles of mRNA  fold change and p value were calculated based on FPKM For MeRIP seq: To identify the methylated sites on RNAs peaks  MACS software was utilized. Differentially methylated sites were identified by diffReps. The motif predictions of MeRIP Seq data were performed by utilizing a Perl script  findMotifGenome.pl  from HOMER software. Genome build: GRCz11 Supplementary files format and content: Gene expression valuesFPKM and Counts and methylated sites,zebrafish brain tissue,Adult wild type zebrafish Danio rerio were bred according to standard methods. For hypoxia treatment  we followed settings established in a previously published protocolYu et al 2011 with modified conditions. Briefly  zebrafish were transferred into a 1 L chamber containing 800 mL predeoxidated water O2: 0.3 0.4 mg/L. Oxygen in the water was then exhausted through the application of 8 L/min nitrogen until the dissolved oxygen value in the water was approximately 0.4 0.6 mg/L. Nitrogen 1 L/min was used to maintain the dissolved oxygen level in the water. post approximately 5 minutes  when the fish became motionless  the zebrafish brain were dissected on ice post anesthetizing to obtain brain tissues. The brain tissues were stored for further analysis in liquid nitrogen.,Ribo Zero rRNA Removal Kits Illumina  San Diego  CA  USA For RNA seq : TruSeq Stranded Total RNA Library Prep Kit Illumina  San Diego  CA  USA For MeRIP seq: GenSeqTM RNA IP Kit GenSeq Inc.  China and NEBNext® Ultra II Directional RNA Library Prep Kit New England Biolabs  Inc.  USA RNA seq and MeRIP seq,,tissue:brain,GSM5832275,GSM5832275: m1A Normoxia IP; Danio rerio; OTHER,GSM5832275 r1,GSM5832275,1,Ribo Zero rRNA Removal Kits Illumina  San Diego  CA  USA For RNA seq : TruSeq Stranded Total RNA Library Prep Kit Illumina  San Diego  CA  USA For MeRIP seq: GenSeqTM RNA IP Kit GenSeq Inc.  China and NEBNext® Ultra II Directional RNA Library Prep Kit New England Biolabs  Inc.  USA RNA seq and MeRIP seq,,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP356476,,loader:fastq load.py,m1A_Normoxia_IP_R1.fastq.gz m1A_Normoxia_IP_R2.fastq.gz,fastq fastq,6882734400.0,22942448.0,GSM5832275 r1,0:150 1:150,A:1321946139;C:1977632664;G:2310916241;T:1272200359;N:38997,150,150,,,1321946139,1977632664,2310916241,1272200359,38997,SRX13883464,SRS11752234,SRA1361294,Affiliated Hospital of Guangdong Medical University,Affiliated Hospital of Guangdong Medical University,2,0.88367,0.88951,0.09774,0.09525,0.86395,0.86401,0.70125,0.74894,150,150,B,B,biological fallback assumption,illumina,novaseq_era,full_length,rrna_depletion,nebnext,bulk,bulk,bulk,,China,2022-01-24,Adult,Adult,Brain,Nervous System
68395,SRR17720622,SRX13883463,SRS11752232,SRP356476,PRJNA800053,Mapping the m1A  m5C  m6A and m7G Methylation Atlas in Zebrafish Brains under Hypoxic Conditions by MeRIP seq,GSE194284,Other,The epigenetic modifications play important regulatory roles in tissue development  maintenance of physiological functions and pathological process. RNA methylations  including newly identified m1A  m5C  m6A and m7G  are important epigenetic modifications. However  how these modifications are distributed in the transcriptome of vertebrate brains and whether their abundance is altered under pathological conditions are still poorly understood. In this study  we chose the model animal of zebrafish to conduct a systematic study to investigate the mRNA methylation atlas in the brain. By performing unbiased analyses of the m1A  m5C  m6A and m7G methylation of mRNA  we found that within the whole brain transcriptome  with the increase of the gene expression levels  the overall level of each of these four modifications on the related genes was also progressively increased. Further bioinformatics analysis indicated that the zebrafish brain has an abundance of m1A modifications. In the hypoxia treated zebrafish brains  the proportion of m1A is decreased  affecting the RNA splicing and zebrafish endogenous retroviruses. Our study presents the first comprehensive atlas of m1A  m5C  m6A and m7G in the epitranscriptome of the zebrafish brain and reveals the distribution of these modifications in mRNA under hypoxic conditions. These data provide an invaluable resource for further research on the involvement of m1A  m5C  m6A and m7G in the regulation of miRNA and repeat elements in vertebrates  and provide new thoughts to study the brain hypoxic injury on the aspect of epitranscriptome. Overall design: For each normoxia control and hypoxia experimental group  10 adult male zebrafish 3 mpf 4 mpf were chosen for the analyses. Three repeats of each experiment were performed and totally 30 fishes per group were collected. The brain tissues around 0.02 g per brain were then stored for further analysis in liquid nitrogen. For each analysis m1A  m5C  m6A  m7G and RNA Seq  the normoxia group and hypoxic group each consisted of 30 mixed brain tissues were analyzed parallelly by RNA seq and MeRIP Seq.,,pubmed:35135476,,m1A Normoxia Input,GSM5832274,,source name:zebrafish brain tissue|tissue:brain,m1A Normoxia Input,Paired end reads were harvested from an Illumina NovaSeq 6000 sequencer Cutadapt v1.9.3  command line software  was used to identify and trim 3’ adapter and low quality bases for rawdata. The clean data were mapped to the reference genome GRCz11 using Hisat2 software v2.0.4. For RNA seq: Guiding by the Ensembl GTF gene annotation file  Cuffdiff software part of cufflinks was used to obtain the gene level FPKM as the expression profiles of mRNA  fold change and p value were calculated based on FPKM For MeRIP seq: To identify the methylated sites on RNAs peaks  MACS software was utilized. Differentially methylated sites were identified by diffReps. The motif predictions of MeRIP Seq data were performed by utilizing a Perl script  findMotifGenome.pl  from HOMER software. Genome build: GRCz11 Supplementary files format and content: Gene expression valuesFPKM and Counts and methylated sites,zebrafish brain tissue,Adult wild type zebrafish Danio rerio were bred according to standard methods. For hypoxia treatment  we followed settings established in a previously published protocolYu et al 2011 with modified conditions. Briefly  zebrafish were transferred into a 1 L chamber containing 800 mL predeoxidated water O2: 0.3 0.4 mg/L. Oxygen in the water was then exhausted through the application of 8 L/min nitrogen until the dissolved oxygen value in the water was approximately 0.4 0.6 mg/L. Nitrogen 1 L/min was used to maintain the dissolved oxygen level in the water. post approximately 5 minutes  when the fish became motionless  the zebrafish brain were dissected on ice post anesthetizing to obtain brain tissues. The brain tissues were stored for further analysis in liquid nitrogen.,Ribo Zero rRNA Removal Kits Illumina  San Diego  CA  USA For RNA seq : TruSeq Stranded Total RNA Library Prep Kit Illumina  San Diego  CA  USA For MeRIP seq: GenSeqTM RNA IP Kit GenSeq Inc.  China and NEBNext® Ultra II Directional RNA Library Prep Kit New England Biolabs  Inc.  USA RNA seq and MeRIP seq,,tissue:brain,GSM5832274,GSM5832274: m1A Normoxia Input; Danio rerio; OTHER,GSM5832274 r1,GSM5832274,1,Ribo Zero rRNA Removal Kits Illumina  San Diego  CA  USA For RNA seq : TruSeq Stranded Total RNA Library Prep Kit Illumina  San Diego  CA  USA For MeRIP seq: GenSeqTM RNA IP Kit GenSeq Inc.  China and NEBNext® Ultra II Directional RNA Library Prep Kit New England Biolabs  Inc.  USA RNA seq and MeRIP seq,,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP356476,,loader:fastq load.py,m1A_Normoxia_Input_R1.fastq.gz m1A_Normoxia_Input_R2.fastq.gz,fastq fastq,7374452700.0,24581509.0,GSM5832274 r1,0:150 1:150,A:1511389847;C:2082118937;G:2325922232;T:1454979400;N:42284,150,150,,,1511389847,2082118937,2325922232,1454979400,42284,SRX13883463,SRS11752232,SRA1361294,Affiliated Hospital of Guangdong Medical University,Affiliated Hospital of Guangdong Medical University,2,0.94589,0.94385,0.18226,0.18222,0.80878,0.8102,0.73686,0.71921,150,150,B,B,biological fallback assumption,illumina,novaseq_era,full_length,rrna_depletion,nebnext,bulk,bulk,bulk,,China,2022-01-24,Adult,Adult,Brain,Nervous System
68396,SRR17720623,SRX13883462,SRS11752231,SRP356476,PRJNA800053,Mapping the m1A  m5C  m6A and m7G Methylation Atlas in Zebrafish Brains under Hypoxic Conditions by MeRIP seq,GSE194284,Other,The epigenetic modifications play important regulatory roles in tissue development  maintenance of physiological functions and pathological process. RNA methylations  including newly identified m1A  m5C  m6A and m7G  are important epigenetic modifications. However  how these modifications are distributed in the transcriptome of vertebrate brains and whether their abundance is altered under pathological conditions are still poorly understood. In this study  we chose the model animal of zebrafish to conduct a systematic study to investigate the mRNA methylation atlas in the brain. By performing unbiased analyses of the m1A  m5C  m6A and m7G methylation of mRNA  we found that within the whole brain transcriptome  with the increase of the gene expression levels  the overall level of each of these four modifications on the related genes was also progressively increased. Further bioinformatics analysis indicated that the zebrafish brain has an abundance of m1A modifications. In the hypoxia treated zebrafish brains  the proportion of m1A is decreased  affecting the RNA splicing and zebrafish endogenous retroviruses. Our study presents the first comprehensive atlas of m1A  m5C  m6A and m7G in the epitranscriptome of the zebrafish brain and reveals the distribution of these modifications in mRNA under hypoxic conditions. These data provide an invaluable resource for further research on the involvement of m1A  m5C  m6A and m7G in the regulation of miRNA and repeat elements in vertebrates  and provide new thoughts to study the brain hypoxic injury on the aspect of epitranscriptome. Overall design: For each normoxia control and hypoxia experimental group  10 adult male zebrafish 3 mpf 4 mpf were chosen for the analyses. Three repeats of each experiment were performed and totally 30 fishes per group were collected. The brain tissues around 0.02 g per brain were then stored for further analysis in liquid nitrogen. For each analysis m1A  m5C  m6A  m7G and RNA Seq  the normoxia group and hypoxic group each consisted of 30 mixed brain tissues were analyzed parallelly by RNA seq and MeRIP Seq.,,pubmed:35135476,,m1A Hypoxia IP,GSM5832273,,source name:zebrafish brain tissue|tissue:brain,m1A Hypoxia IP,Paired end reads were harvested from an Illumina NovaSeq 6000 sequencer Cutadapt v1.9.3  command line software  was used to identify and trim 3’ adapter and low quality bases for rawdata. The clean data were mapped to the reference genome GRCz11 using Hisat2 software v2.0.4. For RNA seq: Guiding by the Ensembl GTF gene annotation file  Cuffdiff software part of cufflinks was used to obtain the gene level FPKM as the expression profiles of mRNA  fold change and p value were calculated based on FPKM For MeRIP seq: To identify the methylated sites on RNAs peaks  MACS software was utilized. Differentially methylated sites were identified by diffReps. The motif predictions of MeRIP Seq data were performed by utilizing a Perl script  findMotifGenome.pl  from HOMER software. Genome build: GRCz11 Supplementary files format and content: Gene expression valuesFPKM and Counts and methylated sites,zebrafish brain tissue,Adult wild type zebrafish Danio rerio were bred according to standard methods. For hypoxia treatment  we followed settings established in a previously published protocolYu et al 2011 with modified conditions. Briefly  zebrafish were transferred into a 1 L chamber containing 800 mL predeoxidated water O2: 0.3 0.4 mg/L. Oxygen in the water was then exhausted through the application of 8 L/min nitrogen until the dissolved oxygen value in the water was approximately 0.4 0.6 mg/L. Nitrogen 1 L/min was used to maintain the dissolved oxygen level in the water. post approximately 5 minutes  when the fish became motionless  the zebrafish brain were dissected on ice post anesthetizing to obtain brain tissues. The brain tissues were stored for further analysis in liquid nitrogen.,Ribo Zero rRNA Removal Kits Illumina  San Diego  CA  USA For RNA seq : TruSeq Stranded Total RNA Library Prep Kit Illumina  San Diego  CA  USA For MeRIP seq: GenSeqTM RNA IP Kit GenSeq Inc.  China and NEBNext® Ultra II Directional RNA Library Prep Kit New England Biolabs  Inc.  USA RNA seq and MeRIP seq,,tissue:brain,GSM5832273,GSM5832273: m1A Hypoxia IP; Danio rerio; OTHER,GSM5832273 r1,GSM5832273,1,Ribo Zero rRNA Removal Kits Illumina  San Diego  CA  USA For RNA seq : TruSeq Stranded Total RNA Library Prep Kit Illumina  San Diego  CA  USA For MeRIP seq: GenSeqTM RNA IP Kit GenSeq Inc.  China and NEBNext® Ultra II Directional RNA Library Prep Kit New England Biolabs  Inc.  USA RNA seq and MeRIP seq,,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP356476,,loader:fastq load.py,m1A_Hypoxia_IP_R1.fastq.gz m1A_Hypoxia_IP_R2.fastq.gz,fastq fastq,4861573800.0,16205246.0,GSM5832273 r1,0:150 1:150,A:927063480;C:1355342107;G:1698345464;T:880801957;N:20792,150,150,,,927063480,1355342107,1698345464,880801957,20792,SRX13883462,SRS11752231,SRA1361294,Affiliated Hospital of Guangdong Medical University,Affiliated Hospital of Guangdong Medical University,2,0.8575,0.86165,0.10013,0.09909,0.87249,0.87221,0.72225,0.66629,150,150,B,B,biological fallback assumption,illumina,novaseq_era,full_length,rrna_depletion,nebnext,bulk,bulk,bulk,,China,2022-01-24,Adult,Adult,Brain,Nervous System
68397,SRR17720624,SRX13883461,SRS11752229,SRP356476,PRJNA800053,Mapping the m1A  m5C  m6A and m7G Methylation Atlas in Zebrafish Brains under Hypoxic Conditions by MeRIP seq,GSE194284,Other,The epigenetic modifications play important regulatory roles in tissue development  maintenance of physiological functions and pathological process. RNA methylations  including newly identified m1A  m5C  m6A and m7G  are important epigenetic modifications. However  how these modifications are distributed in the transcriptome of vertebrate brains and whether their abundance is altered under pathological conditions are still poorly understood. In this study  we chose the model animal of zebrafish to conduct a systematic study to investigate the mRNA methylation atlas in the brain. By performing unbiased analyses of the m1A  m5C  m6A and m7G methylation of mRNA  we found that within the whole brain transcriptome  with the increase of the gene expression levels  the overall level of each of these four modifications on the related genes was also progressively increased. Further bioinformatics analysis indicated that the zebrafish brain has an abundance of m1A modifications. In the hypoxia treated zebrafish brains  the proportion of m1A is decreased  affecting the RNA splicing and zebrafish endogenous retroviruses. Our study presents the first comprehensive atlas of m1A  m5C  m6A and m7G in the epitranscriptome of the zebrafish brain and reveals the distribution of these modifications in mRNA under hypoxic conditions. These data provide an invaluable resource for further research on the involvement of m1A  m5C  m6A and m7G in the regulation of miRNA and repeat elements in vertebrates  and provide new thoughts to study the brain hypoxic injury on the aspect of epitranscriptome. Overall design: For each normoxia control and hypoxia experimental group  10 adult male zebrafish 3 mpf 4 mpf were chosen for the analyses. Three repeats of each experiment were performed and totally 30 fishes per group were collected. The brain tissues around 0.02 g per brain were then stored for further analysis in liquid nitrogen. For each analysis m1A  m5C  m6A  m7G and RNA Seq  the normoxia group and hypoxic group each consisted of 30 mixed brain tissues were analyzed parallelly by RNA seq and MeRIP Seq.,,pubmed:35135476,,m1A Hypoxia Input,GSM5832272,,source name:zebrafish brain tissue|tissue:brain,m1A Hypoxia Input,Paired end reads were harvested from an Illumina NovaSeq 6000 sequencer Cutadapt v1.9.3  command line software  was used to identify and trim 3’ adapter and low quality bases for rawdata. The clean data were mapped to the reference genome GRCz11 using Hisat2 software v2.0.4. For RNA seq: Guiding by the Ensembl GTF gene annotation file  Cuffdiff software part of cufflinks was used to obtain the gene level FPKM as the expression profiles of mRNA  fold change and p value were calculated based on FPKM For MeRIP seq: To identify the methylated sites on RNAs peaks  MACS software was utilized. Differentially methylated sites were identified by diffReps. The motif predictions of MeRIP Seq data were performed by utilizing a Perl script  findMotifGenome.pl  from HOMER software. Genome build: GRCz11 Supplementary files format and content: Gene expression valuesFPKM and Counts and methylated sites,zebrafish brain tissue,Adult wild type zebrafish Danio rerio were bred according to standard methods. For hypoxia treatment  we followed settings established in a previously published protocolYu et al 2011 with modified conditions. Briefly  zebrafish were transferred into a 1 L chamber containing 800 mL predeoxidated water O2: 0.3 0.4 mg/L. Oxygen in the water was then exhausted through the application of 8 L/min nitrogen until the dissolved oxygen value in the water was approximately 0.4 0.6 mg/L. Nitrogen 1 L/min was used to maintain the dissolved oxygen level in the water. post approximately 5 minutes  when the fish became motionless  the zebrafish brain were dissected on ice post anesthetizing to obtain brain tissues. The brain tissues were stored for further analysis in liquid nitrogen.,Ribo Zero rRNA Removal Kits Illumina  San Diego  CA  USA For RNA seq : TruSeq Stranded Total RNA Library Prep Kit Illumina  San Diego  CA  USA For MeRIP seq: GenSeqTM RNA IP Kit GenSeq Inc.  China and NEBNext® Ultra II Directional RNA Library Prep Kit New England Biolabs  Inc.  USA RNA seq and MeRIP seq,,tissue:brain,GSM5832272,GSM5832272: m1A Hypoxia Input; Danio rerio; OTHER,GSM5832272 r1,GSM5832272,1,Ribo Zero rRNA Removal Kits Illumina  San Diego  CA  USA For RNA seq : TruSeq Stranded Total RNA Library Prep Kit Illumina  San Diego  CA  USA For MeRIP seq: GenSeqTM RNA IP Kit GenSeq Inc.  China and NEBNext® Ultra II Directional RNA Library Prep Kit New England Biolabs  Inc.  USA RNA seq and MeRIP seq,,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP356476,,loader:fastq load.py,m1A_Hypoxia_Input_R1.fastq.gz m1A_Hypoxia_Input_R2.fastq.gz,fastq fastq,7262130600.0,24207102.0,GSM5832272 r1,0:150 1:150,A:1553326396;C:2003023914;G:2214285414;T:1491453374;N:41502,150,150,,,1553326396,2003023914,2214285414,1491453374,41502,SRX13883461,SRS11752229,SRA1361294,Affiliated Hospital of Guangdong Medical University,Affiliated Hospital of Guangdong Medical University,2,0.94247,0.94013,0.20625,0.20507,0.80188,0.80137,0.70188,0.73891,150,150,B,B,biological fallback assumption,illumina,novaseq_era,full_length,rrna_depletion,nebnext,bulk,bulk,bulk,,China,2022-01-24,Adult,Adult,Brain,Nervous System
68502,SRR18074848,SRX14226378,SRS12047703,SRP358067,PRJNA803151,mRNA aging shapes the Cap2 methylome in mammalian mRNA,GSE196043,Other,The mRNA cap structure is a major site of dynamic mRNA methylation.  mRNA caps exist in either the Cap1 or Cap2 form  depending on the presence of 2' O methylation on the first  or both the first and second transcribed nucleotide  respectively.  However  the identity of Cap2 containing mRNAs and the function of Cap2 are unclear.  Here we describe CLAM Cap Seq  a method for transcriptome wide mapping and quantification of Cap2.  We find that unlike other epitranscriptomic modifications  Cap2 can occur on all mRNAs.  Cap2 is formed through a slow continuous conversion of mRNAs from Cap1 to Cap2 as mRNAs age in the cytosol.  As a result  Cap2 is enriched on long lived mRNAs.  We find that large increases in the abundance of Cap1 leads to activation of RIG I  especially in conditions where RIG I expression is increased.  The methylation of Cap1 to Cap2 markedly reduces the ability of RNAs to bind and activate RIG I.  We find that the slow Cap2 methylation rate allows Cap2 to accumulate on host mRNAs yet ensures that low Cap2 levels occur on newly expressed viral RNAs.  Overall  these results reveal an immunostimulatory role for Cap1  and that Cap2 functions to reduce activation of the innate immune response. Overall design: We developed next generation sequencing methods for quantification of Cap2 methylation in bulk mRNA CapTag seq  as well as in individual mRNAs in a transcriptome wide manner CLAM Cap seq. Furthermore  we developed tools for measuring translation and stability of transcript start nucleotide TSN mRNA isoforms by combining polysome profiling and actinomycin D treatment with TSS seq transcription start site sequencing.,,pubmed:36725932,,Zebrafish CapQuant seq 2,GSM5907184,,source name:48 hpf embryo|cell line/tissue:whole|genotype:wild type A/B|purification:oligodT25 beads,Zebrafish CapQuant seq 2,Removal of low quality reads and three prime end adapter trimming was performed with flexbar v2.5. Removal of duplicated reads was accomplished with pyFastqDuplicateRemover.py within the pyCRAC package v1.3.2. Read trimming was performed using seqtk package v1.3. Read alignement to fly  mouse or human genome was conducted using Bowtie v1.2.3. five prime end read coverage was calculated per each genomic position using BEDTools v2.28.0. Genome build: Drosohila melanogaster; dm6  Mus musculus; mm10  Homo sapiens; hg38.,48 hpf embryo,,CapTag seq Total RNA was extracted using TRIzol reagent according to the manufacturers instructions followed by isopropanol precipitation.  PolyA+ RNA was isolated from total RNA using magnetic oligodT25 beads NEB.,Zebrafish AB line embryos were maintained in E3 medium at 28 degrees and staged as previously described Kimmel et al.  Developmental Dynamics  1995.,cell line/tissue:whole|genotype:wild type A/B|purification:oligodT25 beads,GSM5907184,GSM5907184: Zebrafish CapQuant seq 2; Danio rerio; OTHER,GSM5907184 r1,GSM5907184,1,CapTag seq Total RNA was extracted using TRIzol reagent according to the manufacturers instructions followed by isopropanol precipitation.  PolyA+ RNA was isolated from total RNA using magnetic oligodT25 beads NEB.,,OTHER,TRANSCRIPTOMIC,other,SINGLE,ILLUMINA,Illumina HiSeq 2500,,SRP358067,,,Zebrafish_CQS2_R1.fastq.gz,fastq,658260009.0,12907059.0,GSM5907184 r1,0:51 1:0,A:158463141;C:142984303;G:213148834;T:143656235;N:7496,51,0,,,158463141,142984303,213148834,143656235,7496,SRX14226378,SRS12047703,SRA1545466,"Pharmacology, Weill Cornell Medicine","Pharmacology, Weill Cornell Medicine",1,0.0,,0.0,,1.0,,,,51,,T,,under 1.2% mapping rate,illumina,hiseq_era,5prime,poly_a,unknown,bulk,bulk,bulk,,United States,2022-02-18,Hatching,Embryo,Embryo Imprecise,All anatomical structures
68503,SRR18074849,SRX14226377,SRS12047702,SRP358067,PRJNA803151,mRNA aging shapes the Cap2 methylome in mammalian mRNA,GSE196043,Other,The mRNA cap structure is a major site of dynamic mRNA methylation.  mRNA caps exist in either the Cap1 or Cap2 form  depending on the presence of 2' O methylation on the first  or both the first and second transcribed nucleotide  respectively.  However  the identity of Cap2 containing mRNAs and the function of Cap2 are unclear.  Here we describe CLAM Cap Seq  a method for transcriptome wide mapping and quantification of Cap2.  We find that unlike other epitranscriptomic modifications  Cap2 can occur on all mRNAs.  Cap2 is formed through a slow continuous conversion of mRNAs from Cap1 to Cap2 as mRNAs age in the cytosol.  As a result  Cap2 is enriched on long lived mRNAs.  We find that large increases in the abundance of Cap1 leads to activation of RIG I  especially in conditions where RIG I expression is increased.  The methylation of Cap1 to Cap2 markedly reduces the ability of RNAs to bind and activate RIG I.  We find that the slow Cap2 methylation rate allows Cap2 to accumulate on host mRNAs yet ensures that low Cap2 levels occur on newly expressed viral RNAs.  Overall  these results reveal an immunostimulatory role for Cap1  and that Cap2 functions to reduce activation of the innate immune response. Overall design: We developed next generation sequencing methods for quantification of Cap2 methylation in bulk mRNA CapTag seq  as well as in individual mRNAs in a transcriptome wide manner CLAM Cap seq. Furthermore  we developed tools for measuring translation and stability of transcript start nucleotide TSN mRNA isoforms by combining polysome profiling and actinomycin D treatment with TSS seq transcription start site sequencing.,,pubmed:36725932,,Zebrafish CapQuant seq 1,GSM5907183,,source name:48 hpf embryo|cell line/tissue:whole|genotype:wild type A/B|purification:oligodT25 beads,Zebrafish CapQuant seq 1,Removal of low quality reads and three prime end adapter trimming was performed with flexbar v2.5. Removal of duplicated reads was accomplished with pyFastqDuplicateRemover.py within the pyCRAC package v1.3.2. Read trimming was performed using seqtk package v1.3. Read alignement to fly  mouse or human genome was conducted using Bowtie v1.2.3. five prime end read coverage was calculated per each genomic position using BEDTools v2.28.0. Genome build: Drosohila melanogaster; dm6  Mus musculus; mm10  Homo sapiens; hg38.,48 hpf embryo,,CapTag seq Total RNA was extracted using TRIzol reagent according to the manufacturers instructions followed by isopropanol precipitation.  PolyA+ RNA was isolated from total RNA using magnetic oligodT25 beads NEB.,Zebrafish AB line embryos were maintained and staged as previously described Kimmel et al.  Developmental Dynamics  1995.,cell line/tissue:whole|genotype:wild type A/B|purification:oligodT25 beads,GSM5907183,GSM5907183: Zebrafish CapQuant seq 1; Danio rerio; OTHER,GSM5907183 r1,GSM5907183,1,CapTag seq Total RNA was extracted using TRIzol reagent according to the manufacturers instructions followed by isopropanol precipitation.  PolyA+ RNA was isolated from total RNA using magnetic oligodT25 beads NEB.,,OTHER,TRANSCRIPTOMIC,other,SINGLE,ILLUMINA,Illumina HiSeq 2500,,SRP358067,,,Zebrafish_CQS1_R1.fastq.gz,fastq,1636691694.0,32091994.0,GSM5907183 r1,0:51 1:0,A:394187895;C:355520591;G:529426272;T:357424863;N:132073,51,0,,,394187895,355520591,529426272,357424863,132073,SRX14226377,SRS12047702,SRA1545466,"Pharmacology, Weill Cornell Medicine","Pharmacology, Weill Cornell Medicine",1,0.0,,0.0,,1.0,,,,51,,T,,under 1.2% mapping rate,illumina,hiseq_era,5prime,poly_a,unknown,bulk,bulk,bulk,,United States,2022-02-18,Hatching,Embryo,Embryo Imprecise,All anatomical structures
71044,SRR21140654,SRX17152943,SRS14724788,SRP393052,PRJNA871254,Mycn regulates intestinal development through ribosomal biogenesis in a zebrafish model of Feingold syndrome 1 RNA seq and Ribo seq,GSE211652,Other,Purpose: To systematically investigate the molecular mechanisms resulting from Mycn loss of function. Methods:  Approximately 30 mycn mutant or WT zebrafish embryos at 72 hpf were harvested . Libraries were prepared using Chromium Controller and Chromium Single Cell three primeLibrary & Gel Bead Kit v3 10x Genomics  PN 1000074 according to the manufacturer's protocol for 10000 cells recovery. Results: More than 3 000 genes showed reduced tranlation efficiency in Mycn mutant. Conclusions: mTOR signaling was reduced in Mycn mutant. Overall design: wild type or mycn zebrafish embryos at 72 hpf were harvested for RNA seq and Ribo seq.,,,,Mycn mutant zebrafish embryos Ribo seq 72 hpf replicate 2,GSM6482085,,source name:zebrafish cells|strain:Mycn mutant|tissue:embryonic cells|age:72 hpf,Mycn mutant  zebrafish embryos Ribo seq 72 hpf replicate 2,The sequencing reads were aligned to the zebrafish GRCz11 genome using STAR  and reads mapped to multiple genomic location were removed. Gene expression counts of each sample were calculated by featureCounts. Differential expression analysis was performed by limma package. Assembly: GRCz11,zebrafish cells,WT and Mycn mutant zebrafish embryos were collected for RNA sequencing or Ribo seq at 72 hpf,For bulk RNA seq  RNA libraries were prepared for sequencing using standard Illumina protocols. For Ribo seq  WT and mycn mutant embryos were collected at 3 dpf  digested with cold 0.5% trypsin to a single cell suspension  and homogenized in lysis buffer for 20–50 times. The homogenate centrifuged at 12 000 g for 15 min at 4°C  and the supernatants were digested with RNase for 30 mins and aborted by 1M EGTA followed by layering on top of a 10%–50% sucrose gradient solution. Ultracentrifugation was performed at 36 000 rpm for 2 hrs hours at 4°C. post centrifugation  the fractions were collected according to the absorbance at optical density OD260 by a TRAIX detector. Then the RNA was purified and used to construct the library for sequencing.,embryos were cultured in a Petri dish filled with 0.3X Danieau buffer,strain:Mycn mutant|tissue:embryonic cells|age:72 hpf,GSM6482085,GSM6482085: Mycn mutant  zebrafish embryos Ribo seq 72 hpf replicate 2; Danio rerio; OTHER,GSM6482085 r1,GSM6482085,1,For bulk RNA seq  RNA libraries were prepared for sequencing using standard Illumina protocols. For Ribo seq  WT and mycn mutant embryos were collected at 3 dpf  digested with cold 0.5% trypsin to a single cell suspension  and homogenized in lysis buffer for 20–50 times. The homogenate centrifuged at 12 000 g for 15 min at 4°C  and the supernatants were digested with RNase for 30 mins and aborted by 1M EGTA followed by layering on top of a 10%–50% sucrose gradient solution. Ultracentrifugation was performed at 36 000 rpm for 2 hrs hours at 4°C. post centrifugation  the fractions were collected according to the absorbance at optical density OD260 by a TRAIX detector. Then the RNA was purified and used to construct the library for sequencing.,,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP393052,,,mut2-riboseq_1.clean.fq.gz mut2-riboseq_2.clean.fq.gz,fastq fastq,405742417.0,6982873.0,GSM6482085 r1,0:29.04 1:29.07,A:98515642;C:104430844;G:104419248;T:98371492;N:5191,29,29,,,98515642,104430844,104419248,98371492,5191,SRX17152943,SRS14724788,SRA1479247,"Institute of genetics, Zhejiang University","Institute of genetics, Zhejiang University",2,0.4308,0.42905,0.15957,0.1591,0.74292,0.74302,0.56011,0.55773,40,40,B,B,biological fallback assumption,illumina,novaseq_era,unknown,other,unknown,sc,bulk,bulk,,China,2022-08-19,Larval,Larval,Embryo Imprecise,All anatomical structures
71045,SRR21140655,SRX17152942,SRS14724787,SRP393052,PRJNA871254,Mycn regulates intestinal development through ribosomal biogenesis in a zebrafish model of Feingold syndrome 1 RNA seq and Ribo seq,GSE211652,Other,Purpose: To systematically investigate the molecular mechanisms resulting from Mycn loss of function. Methods:  Approximately 30 mycn mutant or WT zebrafish embryos at 72 hpf were harvested . Libraries were prepared using Chromium Controller and Chromium Single Cell three primeLibrary & Gel Bead Kit v3 10x Genomics  PN 1000074 according to the manufacturer's protocol for 10000 cells recovery. Results: More than 3 000 genes showed reduced tranlation efficiency in Mycn mutant. Conclusions: mTOR signaling was reduced in Mycn mutant. Overall design: wild type or mycn zebrafish embryos at 72 hpf were harvested for RNA seq and Ribo seq.,,,,Mycn mutant zebrafish embryos Ribo seq 72 hpf replicate 1,GSM6482084,,source name:zebrafish cells|strain:Mycn mutant|tissue:embryonic cells|age:72 hpf,Mycn mutant  zebrafish embryos Ribo seq 72 hpf replicate 1,The sequencing reads were aligned to the zebrafish GRCz11 genome using STAR  and reads mapped to multiple genomic location were removed. Gene expression counts of each sample were calculated by featureCounts. Differential expression analysis was performed by limma package. Assembly: GRCz11,zebrafish cells,WT and Mycn mutant zebrafish embryos were collected for RNA sequencing or Ribo seq at 72 hpf,For bulk RNA seq  RNA libraries were prepared for sequencing using standard Illumina protocols. For Ribo seq  WT and mycn mutant embryos were collected at 3 dpf  digested with cold 0.5% trypsin to a single cell suspension  and homogenized in lysis buffer for 20–50 times. The homogenate centrifuged at 12 000 g for 15 min at 4°C  and the supernatants were digested with RNase for 30 mins and aborted by 1M EGTA followed by layering on top of a 10%–50% sucrose gradient solution. Ultracentrifugation was performed at 36 000 rpm for 2 hrs hours at 4°C. post centrifugation  the fractions were collected according to the absorbance at optical density OD260 by a TRAIX detector. Then the RNA was purified and used to construct the library for sequencing.,embryos were cultured in a Petri dish filled with 0.3X Danieau buffer,strain:Mycn mutant|tissue:embryonic cells|age:72 hpf,GSM6482084,GSM6482084: Mycn mutant  zebrafish embryos Ribo seq 72 hpf replicate 1; Danio rerio; OTHER,GSM6482084 r1,GSM6482084,1,For bulk RNA seq  RNA libraries were prepared for sequencing using standard Illumina protocols. For Ribo seq  WT and mycn mutant embryos were collected at 3 dpf  digested with cold 0.5% trypsin to a single cell suspension  and homogenized in lysis buffer for 20–50 times. The homogenate centrifuged at 12 000 g for 15 min at 4°C  and the supernatants were digested with RNase for 30 mins and aborted by 1M EGTA followed by layering on top of a 10%–50% sucrose gradient solution. Ultracentrifugation was performed at 36 000 rpm for 2 hrs hours at 4°C. post centrifugation  the fractions were collected according to the absorbance at optical density OD260 by a TRAIX detector. Then the RNA was purified and used to construct the library for sequencing.,,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP393052,,,mut1-riboseq_1.clean.fq.gz mut1-riboseq_2.clean.fq.gz,fastq fastq,491622744.0,8480542.0,GSM6482084 r1,0:28.97 1:29.01,A:119352696;C:126567605;G:126525090;T:119169287;N:8066,28,29,,,119352696,126567605,126525090,119169287,8066,SRX17152942,SRS14724787,SRA1479247,"Institute of genetics, Zhejiang University","Institute of genetics, Zhejiang University",2,0.43508,0.4326,0.15983,0.15894,0.7388,0.73939,0.55997,0.56089,30,30,B,B,biological fallback assumption,illumina,novaseq_era,unknown,other,unknown,sc,bulk,bulk,,China,2022-08-19,Larval,Larval,Embryo Imprecise,All anatomical structures
71046,SRR21140656,SRX17152941,SRS14724786,SRP393052,PRJNA871254,Mycn regulates intestinal development through ribosomal biogenesis in a zebrafish model of Feingold syndrome 1 RNA seq and Ribo seq,GSE211652,Other,Purpose: To systematically investigate the molecular mechanisms resulting from Mycn loss of function. Methods:  Approximately 30 mycn mutant or WT zebrafish embryos at 72 hpf were harvested . Libraries were prepared using Chromium Controller and Chromium Single Cell three primeLibrary & Gel Bead Kit v3 10x Genomics  PN 1000074 according to the manufacturer's protocol for 10000 cells recovery. Results: More than 3 000 genes showed reduced tranlation efficiency in Mycn mutant. Conclusions: mTOR signaling was reduced in Mycn mutant. Overall design: wild type or mycn zebrafish embryos at 72 hpf were harvested for RNA seq and Ribo seq.,,,,wild type zebrafish embryos Ribo seq 72 hpf replicate 2,GSM6482083,,source name:zebrafish cells|strain:AB|tissue:embryonic cells|age:72 hpf,wild type  zebrafish embryos Ribo seq 72 hpf replicate 2,The sequencing reads were aligned to the zebrafish GRCz11 genome using STAR  and reads mapped to multiple genomic location were removed. Gene expression counts of each sample were calculated by featureCounts. Differential expression analysis was performed by limma package. Assembly: GRCz11,zebrafish cells,WT and Mycn mutant zebrafish embryos were collected for RNA sequencing or Ribo seq at 72 hpf,For bulk RNA seq  RNA libraries were prepared for sequencing using standard Illumina protocols. For Ribo seq  WT and mycn mutant embryos were collected at 3 dpf  digested with cold 0.5% trypsin to a single cell suspension  and homogenized in lysis buffer for 20–50 times. The homogenate centrifuged at 12 000 g for 15 min at 4°C  and the supernatants were digested with RNase for 30 mins and aborted by 1M EGTA followed by layering on top of a 10%–50% sucrose gradient solution. Ultracentrifugation was performed at 36 000 rpm for 2 hrs hours at 4°C. post centrifugation  the fractions were collected according to the absorbance at optical density OD260 by a TRAIX detector. Then the RNA was purified and used to construct the library for sequencing.,embryos were cultured in a Petri dish filled with 0.3X Danieau buffer,strain:AB|tissue:embryonic cells|age:72 hpf,GSM6482083,GSM6482083: wild type  zebrafish embryos Ribo seq 72 hpf replicate 2; Danio rerio; OTHER,GSM6482083 r1,GSM6482083,1,For bulk RNA seq  RNA libraries were prepared for sequencing using standard Illumina protocols. For Ribo seq  WT and mycn mutant embryos were collected at 3 dpf  digested with cold 0.5% trypsin to a single cell suspension  and homogenized in lysis buffer for 20–50 times. The homogenate centrifuged at 12 000 g for 15 min at 4°C  and the supernatants were digested with RNase for 30 mins and aborted by 1M EGTA followed by layering on top of a 10%–50% sucrose gradient solution. Ultracentrifugation was performed at 36 000 rpm for 2 hrs hours at 4°C. post centrifugation  the fractions were collected according to the absorbance at optical density OD260 by a TRAIX detector. Then the RNA was purified and used to construct the library for sequencing.,,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP393052,,,wt2-riboseq_1.clean.fq.gz wt2-riboseq_2.clean.fq.gz,fastq fastq,509843192.0,8545402.0,GSM6482083 r1,0:29.81 1:29.85,A:121579451;C:133424275;G:133469726;T:121359509;N:10231,29,29,,,121579451,133424275,133469726,121359509,10231,SRX17152941,SRS14724786,SRA1479247,"Institute of genetics, Zhejiang University","Institute of genetics, Zhejiang University",2,0.47779,0.47724,0.17114,0.17128,0.73296,0.73208,0.5655,0.56416,23,23,B,B,biological fallback assumption,illumina,novaseq_era,unknown,other,unknown,sc,bulk,bulk,,China,2022-08-19,Larval,Larval,Embryo Imprecise,All anatomical structures
71047,SRR21140657,SRX17152940,SRS14724785,SRP393052,PRJNA871254,Mycn regulates intestinal development through ribosomal biogenesis in a zebrafish model of Feingold syndrome 1 RNA seq and Ribo seq,GSE211652,Other,Purpose: To systematically investigate the molecular mechanisms resulting from Mycn loss of function. Methods:  Approximately 30 mycn mutant or WT zebrafish embryos at 72 hpf were harvested . Libraries were prepared using Chromium Controller and Chromium Single Cell three primeLibrary & Gel Bead Kit v3 10x Genomics  PN 1000074 according to the manufacturer's protocol for 10000 cells recovery. Results: More than 3 000 genes showed reduced tranlation efficiency in Mycn mutant. Conclusions: mTOR signaling was reduced in Mycn mutant. Overall design: wild type or mycn zebrafish embryos at 72 hpf were harvested for RNA seq and Ribo seq.,,,,wild type zebrafish embryos Ribo seq 72 hpf replicate 1,GSM6482082,,source name:zebrafish cells|strain:AB|tissue:embryonic cells|age:72 hpf,wild type  zebrafish embryos Ribo seq 72 hpf replicate 1,The sequencing reads were aligned to the zebrafish GRCz11 genome using STAR  and reads mapped to multiple genomic location were removed. Gene expression counts of each sample were calculated by featureCounts. Differential expression analysis was performed by limma package. Assembly: GRCz11,zebrafish cells,WT and Mycn mutant zebrafish embryos were collected for RNA sequencing or Ribo seq at 72 hpf,For bulk RNA seq  RNA libraries were prepared for sequencing using standard Illumina protocols. For Ribo seq  WT and mycn mutant embryos were collected at 3 dpf  digested with cold 0.5% trypsin to a single cell suspension  and homogenized in lysis buffer for 20–50 times. The homogenate centrifuged at 12 000 g for 15 min at 4°C  and the supernatants were digested with RNase for 30 mins and aborted by 1M EGTA followed by layering on top of a 10%–50% sucrose gradient solution. Ultracentrifugation was performed at 36 000 rpm for 2 hrs hours at 4°C. post centrifugation  the fractions were collected according to the absorbance at optical density OD260 by a TRAIX detector. Then the RNA was purified and used to construct the library for sequencing.,embryos were cultured in a Petri dish filled with 0.3X Danieau buffer,strain:AB|tissue:embryonic cells|age:72 hpf,GSM6482082,GSM6482082: wild type  zebrafish embryos Ribo seq 72 hpf replicate 1; Danio rerio; OTHER,GSM6482082 r1,GSM6482082,1,For bulk RNA seq  RNA libraries were prepared for sequencing using standard Illumina protocols. For Ribo seq  WT and mycn mutant embryos were collected at 3 dpf  digested with cold 0.5% trypsin to a single cell suspension  and homogenized in lysis buffer for 20–50 times. The homogenate centrifuged at 12 000 g for 15 min at 4°C  and the supernatants were digested with RNase for 30 mins and aborted by 1M EGTA followed by layering on top of a 10%–50% sucrose gradient solution. Ultracentrifugation was performed at 36 000 rpm for 2 hrs hours at 4°C. post centrifugation  the fractions were collected according to the absorbance at optical density OD260 by a TRAIX detector. Then the RNA was purified and used to construct the library for sequencing.,,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP393052,,,wt1-riboseq_1.clean.fq.gz wt1-riboseq_2.clean.fq.gz,fastq fastq,704869055.0,11792221.0,GSM6482082 r1,0:29.87 1:29.90,A:168078449;C:184483903;G:184537174;T:167755786;N:13743,29,29,,,168078449,184483903,184537174,167755786,13743,SRX17152940,SRS14724785,SRA1479247,"Institute of genetics, Zhejiang University","Institute of genetics, Zhejiang University",2,0.4713,0.46871,0.16916,0.16911,0.73746,0.73843,0.56441,0.56008,22,22,B,B,biological fallback assumption,illumina,novaseq_era,unknown,other,unknown,sc,bulk,bulk,,China,2022-08-19,Larval,Larval,Embryo Imprecise,All anatomical structures