{"database": "metadata", "table": "run_metadata", "rows": [[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.", null, null, null, "Fish embryo mRNA 1024cell PAL seq v4", "GSM7716868", null, "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 \u201c m 15   quality base=64  q 20 20   match read wildcards  e 0.05  a NNNNATCTCGTATGCCGTCTTCTGCTTG  O 7\u201d. 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 \u201c  runThreadN 16   runMode alignReads   outFilterMultimapNmax 1   outReadsUnmapped Fastx   outFilterType BySJout   outSAMattributes All   outSAMtype BAM Unsorted SortedByCoordinate\u201d. 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\u20137 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", null, "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 \u00b5g/ml cycloheximide  cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer]  and 200 units/ml SUPERase\u2022In in a volume of 10 \u00b5l per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4\u00b0C 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\u00b0C 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 3\u02b9 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 \u00b5l. The RNA and oligos were annealed by incubation at 70\u00b0C for 5 min and then slowly cooling to 23\u00b0C at 0.1\u00b0C/sec. The annealed mixture was combined with 40 \u00b5l MyOne Streptavidin C1 beads Thermo Fisher  65002 and incubated for 20 min at 23\u00b0C 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 \u00b5l 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 \u00b5l 2x SSC. The RNA was eluted from the beads first with 100 \u00b5l 10 mM HEPES pH 7.5 at 65\u00b0C for 3 min and then second with 100 \u00b5l water at 65\u00b0C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 \u00b5l water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3\u02b9 adapter in a 10 \u00b5l reaction containing 5 \u00b5M 3\u02b9 adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/\u00b5l T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23\u00b0C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 \u00b5l water. The ligated RNA was mixed with 0.6 \u00b5l 100 \u00b5M reverse transcription primer KXS037 in a total volume of 12 \u00b5l  incubated at 65\u00b0C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 \u00b5l reaction containing 1x First Strand Buffer  500 \u00b5M dNTPs  5 mM dithiothreitol  1 unit/\u00b5l SUPERase\u2022In  and 200 units SuperScript III Thermo Fisher  18080044 at 50\u00b0C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 \u00b5l 1 M NaOH at 90\u00b0C for 10 min  followed by neutralization with 36.7 \u00b5l 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 \u00b5l PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer\u2019s suggested protocol for 10\u201315 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 3\u02b9 adapter KXS013 was used for 3\u02b9 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 \u00b5l. The cDNAs and the oligo were annealed by incubation at 65\u00b0C for 5 min and then slowly cooling to 23\u00b0C at 0.1\u00b0C/sec. The annealed mixture was combined with 100 \u00b5l MyOne Streptavidin C1 beads and incubated for 20 min at 23\u00b0C 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 \u00b5l 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 \u00b5l water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.", null, "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 \u00b5g/ml cycloheximide  cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer]  and 200 units/ml SUPERase\u2022In in a volume of 10 \u00b5l per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4\u00b0C 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\u00b0C 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 3\u02b9 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 \u00b5l. The RNA and oligos were annealed by incubation at 70\u00b0C for 5 min and then slowly cooling to 23\u00b0C at 0.1\u00b0C/sec. The annealed mixture was combined with 40 \u00b5l MyOne Streptavidin C1 beads Thermo Fisher  65002 and incubated for 20 min at 23\u00b0C 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 \u00b5l 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 \u00b5l 2x SSC. The RNA was eluted from the beads first with 100 \u00b5l 10 mM HEPES pH 7.5 at 65\u00b0C for 3 min and then second with 100 \u00b5l water at 65\u00b0C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 \u00b5l water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3\u02b9 adapter in a 10 \u00b5l reaction containing 5 \u00b5M 3\u02b9 adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/\u00b5l T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23\u00b0C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 \u00b5l water. The ligated RNA was mixed with 0.6 \u00b5l 100 \u00b5M reverse transcription primer KXS037 in a total volume of 12 \u00b5l  incubated at 65\u00b0C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 \u00b5l reaction containing 1x First Strand Buffer  500 \u00b5M dNTPs  5 mM dithiothreitol  1 unit/\u00b5l SUPERase\u2022In  and 200 units SuperScript III Thermo Fisher  18080044 at 50\u00b0C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 \u00b5l 1 M NaOH at 90\u00b0C for 10 min  followed by neutralization with 36.7 \u00b5l 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 \u00b5l PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer's suggested protocol for 10\u201315 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 3\u02b9 adapter KXS013 was used for 3\u02b9 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 \u00b5l. The cDNAs and the oligo were annealed by incubation at 65\u00b0C for 5 min and then slowly cooling to 23\u00b0C at 0.1\u00b0C/sec. The annealed mixture was combined with 100 \u00b5l MyOne Streptavidin C1 beads and incubated for 20 min at 23\u00b0C 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 \u00b5l 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 \u00b5l water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "Illumina HiSeq 2500", null, "SRP455680", null, null, "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, null, null, 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", null, "United States", "2023-08-17", "Zygote", "Embryo", "Embryo Imprecise", "All anatomical structures"]], "columns": ["rowid", "run.accession", "experiment.accession", "sample.accession", "study.accession", "bioproject", "study.title", "study.alias", "study.type", "study.abstract", "study.attributes", "study.PMIDs", "sample.description", "sample.title", "sample.alias", "sample.centername", "sample.attributes", "GEOsample.title", "GEOsample.dataprocessing", "GEOsample.source", "GEOsample.treatmentprotocol", "GEOsample.extractprotocol", "GEOsample.growthprotocol", "GEOsample.characteristics", "GEOsample.accession", "experiment.title", "experiment.alias", "experiment.library_name", "experiment.design_description", "experiment.library_construction_protocol", "experiment.attributes", "experiment.library_strategy", "experiment.library_source", "experiment.library_selection", "experiment.library_layout", "experiment.platform", "experiment.instrument_model", "experiment.spot_descriptor", "experiment.study_ref", "run.title", "run.attributes", "run.filename", "run.semantic_name", "run.total_bases", "run.total_spots", "run.alias", "run.read_lengths", "run.base_counts", "run.r1_length", "run.r2_length", "run.r3_length", "run.r4_length", "run.Acount", "run.Ccount", "run.Gcount", "run.Tcount", "run.Ncount", "run.experiment", "run.pool_member", "submission.accession", "submission.srasource", "submission.bioprojectsource", "seqdetective.n_mates", "seqdetective.mapping_rate.mate1", "seqdetective.mapping_rate.mate2", "seqdetective.nofeature_rate.mate1", "seqdetective.nofeature_rate.mate2", "seqdetective.sparsity.mate1", "seqdetective.sparsity.mate2", "seqdetective.pos_strand_rate.mate1", "seqdetective.pos_strand_rate.mate2", "seqdetective.readlen.mate1", "seqdetective.readlen.mate2", "seqdetective.judgement.mate1", "seqdetective.judgement.mate2", "seqdetective.judgement.reason", "platform_family", "instrument_generation", "read_bias", "selection_class", "prep_kit", "sc_or_bulk", "tech_class", "technology", "tech_variant", "submission.bioprojectsource.country", "earliest_date", "devstage_curation", "devstage_curation_coarse", "tissue_curation", "tissue_curation_coarse"], "primary_keys": ["rowid"], "primary_key_values": ["25188"], "units": {}, "query_ms": 8.788947998255026}