run_metadata: 25187

This data as json

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
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