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27 rows where devstage_curation = "Zygote" and experiment.library_strategy = "RIP-Seq"

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technology 4

tissue_curation 3

tissue_curation_coarse 2

experiment.library_layout 2

experiment.platform 1

experiment.library_strategy 1

  • RIP-Seq · 27 ✖

experiment.library_source 1

experiment.library_selection 1

devstage_curation_coarse 1

devstage_curation 1

  • Zygote · 27 ✖
Link rowid ▼ run.accession experiment.accession sample.accession study.accession bioproject study.title study.alias study.type study.abstract study.attributes study.PMIDs sample.description sample.title sample.alias sample.centername sample.attributes GEOsample.title GEOsample.dataprocessing GEOsample.source GEOsample.treatmentprotocol GEOsample.extractprotocol GEOsample.growthprotocol GEOsample.characteristics GEOsample.accession experiment.title experiment.alias experiment.library_name experiment.design_description experiment.library_construction_protocol experiment.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
40701 40701 SRR3231363 SRX1637111 SRS1342926 SRP071849 PRJNA315400 Tdrd6a recruits Ziwi bound piRNAs and coordinates deposition of germ plasm mRNAs into primordial germ cells [RIP Seq] GSE79161 Transcriptome Analysis Germ plasm the Balbiani body and nuage are evolutionary conserved structures essential for germ cell specification and maintenance. We describe Tdrd6a as a component of these structures with two distinct molecular functions. First Tdrd6a facilitates the accumulation of the typical antisense bias of piRNAs without xxx effects on piRNA biogenesis signatures. Second we show that Tdrd6a is required for Balbiani body and germ plasm integrity and associates with RNA binding proteins and germ plasm mRNAs. On the cell biological level maternally contributed Tdrd6a strongly impacts germ cell formation but is dispensable for fertility. Using single cell RNA sequencing we demonstrate that Tdrd6a promotes early germ cell development and regulates the stoichiometry of germ plasm mRNAs. We propose that Tdrd6a functions as a scaffold to recruit correct ratios of germ plasm transcripts and to accumulate antisense piRNA complexes in order to ensure both specification and maintenance of germ cells. Overall design: RNA was extracted from ovary tissue or from RIP experiments by Trizol extraction. parent bioproject:PRJNA315403 pubmed:30086300   Input 3 GSM2087141   tissue:Zebrafish Zygotes|fraction:Input Input 3 Reads were mapped to Zv9 using tophat trapnell et al 2009to the ENSEMBLE gene build and further processing of the read counts was performed using Deseq Anders et al 2009 Genome build: zv9 Supplementary files format and content: The mRNA counts.csv contains the DESeq normalized read counts per Ensemble gene as indicated. Headers indicate the sample name. Zebrafish Zygotes   mRNA was extracted from zygotes or by RIP experiments followed by Trizol extraction The RNA was extracted using chloroform and precipitated with iso propanol. Ovation RNA seq System V2 NuGEN library preperation kit was used and the library was sequenced on an Illumina HISEQ using 50bp single end sequencing. Zebrafish were maintained under standard conditions. fraction:Input GSM2087141 GSM2087141: Input 3; Danio rerio; RIP Seq GSM2087141   1 mRNA was extracted from zygotes or by RIP experiments followed by Trizol extraction The RNA was extracted using chloroform and precipitated with iso propanol. Ovation RNA seq System V2 NuGEN library preperation kit was used and the library was sequenced on an Illumina HISEQ using 50bp single end sequencing. GEO Accession:GSM2087141 RIP-Seq TRANSCRIPTOMIC other SINGLE ILLUMINA Illumina HiSeq 2000   SRP071849     WT_Input_3.fastq.gz fastq 1902296226.0 37299926.0 GSM2087141 r1 0:51 A:466290166;C:465344039;G:486942278;T:483240280;N:479463 51       466290166 465344039 486942278 483240280 479463 SRX1637111 SRS1342926 SRA385813 GEO Rene Ketting, RNA silencing, IMB 1 0.85793   0.14814   0.79941   0.55214   51   B   usable mapping rate illumina hiseq_era unknown other unknown sc_generic single_cell_generic generic-scrnaseq-only   Germany 2016-03-13 Zygote Embryo Oocyte Reproductive System
40702 40702 SRR3231362 SRX1637110 SRS1342927 SRP071849 PRJNA315400 Tdrd6a recruits Ziwi bound piRNAs and coordinates deposition of germ plasm mRNAs into primordial germ cells [RIP Seq] GSE79161 Transcriptome Analysis Germ plasm the Balbiani body and nuage are evolutionary conserved structures essential for germ cell specification and maintenance. We describe Tdrd6a as a component of these structures with two distinct molecular functions. First Tdrd6a facilitates the accumulation of the typical antisense bias of piRNAs without xxx effects on piRNA biogenesis signatures. Second we show that Tdrd6a is required for Balbiani body and germ plasm integrity and associates with RNA binding proteins and germ plasm mRNAs. On the cell biological level maternally contributed Tdrd6a strongly impacts germ cell formation but is dispensable for fertility. Using single cell RNA sequencing we demonstrate that Tdrd6a promotes early germ cell development and regulates the stoichiometry of germ plasm mRNAs. We propose that Tdrd6a functions as a scaffold to recruit correct ratios of germ plasm transcripts and to accumulate antisense piRNA complexes in order to ensure both specification and maintenance of germ cells. Overall design: RNA was extracted from ovary tissue or from RIP experiments by Trizol extraction. parent bioproject:PRJNA315403 pubmed:30086300   Input 2 GSM2087140   tissue:Zebrafish Zygotes|fraction:Input Input 2 Reads were mapped to Zv9 using tophat trapnell et al 2009to the ENSEMBLE gene build and further processing of the read counts was performed using Deseq Anders et al 2009 Genome build: zv9 Supplementary files format and content: The mRNA counts.csv contains the DESeq normalized read counts per Ensemble gene as indicated. Headers indicate the sample name. Zebrafish Zygotes   mRNA was extracted from zygotes or by RIP experiments followed by Trizol extraction The RNA was extracted using chloroform and precipitated with iso propanol. Ovation RNA seq System V2 NuGEN library preperation kit was used and the library was sequenced on an Illumina HISEQ using 50bp single end sequencing. Zebrafish were maintained under standard conditions. fraction:Input GSM2087140 GSM2087140: Input 2; Danio rerio; RIP Seq GSM2087140   1 mRNA was extracted from zygotes or by RIP experiments followed by Trizol extraction The RNA was extracted using chloroform and precipitated with iso propanol. Ovation RNA seq System V2 NuGEN library preperation kit was used and the library was sequenced on an Illumina HISEQ using 50bp single end sequencing. GEO Accession:GSM2087140 RIP-Seq TRANSCRIPTOMIC other SINGLE ILLUMINA Illumina HiSeq 2000   SRP071849     WT_Input_2.fastq.gz fastq 1540737999.0 30210549.0 GSM2087140 r1 0:51 A:380481772;C:379741192;G:395453141;T:384675313;N:386581 51       380481772 379741192 395453141 384675313 386581 SRX1637110 SRS1342927 SRA385813 GEO Rene Ketting, RNA silencing, IMB 1 0.8787   0.14935   0.79202   0.54381   51   B   usable mapping rate illumina hiseq_era unknown other unknown sc_generic single_cell_generic generic-scrnaseq-only   Germany 2016-03-13 Zygote Embryo Oocyte Reproductive System
40703 40703 SRR3231361 SRX1637109 SRS1342928 SRP071849 PRJNA315400 Tdrd6a recruits Ziwi bound piRNAs and coordinates deposition of germ plasm mRNAs into primordial germ cells [RIP Seq] GSE79161 Transcriptome Analysis Germ plasm the Balbiani body and nuage are evolutionary conserved structures essential for germ cell specification and maintenance. We describe Tdrd6a as a component of these structures with two distinct molecular functions. First Tdrd6a facilitates the accumulation of the typical antisense bias of piRNAs without xxx effects on piRNA biogenesis signatures. Second we show that Tdrd6a is required for Balbiani body and germ plasm integrity and associates with RNA binding proteins and germ plasm mRNAs. On the cell biological level maternally contributed Tdrd6a strongly impacts germ cell formation but is dispensable for fertility. Using single cell RNA sequencing we demonstrate that Tdrd6a promotes early germ cell development and regulates the stoichiometry of germ plasm mRNAs. We propose that Tdrd6a functions as a scaffold to recruit correct ratios of germ plasm transcripts and to accumulate antisense piRNA complexes in order to ensure both specification and maintenance of germ cells. Overall design: RNA was extracted from ovary tissue or from RIP experiments by Trizol extraction. parent bioproject:PRJNA315403 pubmed:30086300   Input 1 GSM2087139   tissue:Zebrafish Zygotes|fraction:Input Input 1 Reads were mapped to Zv9 using tophat trapnell et al 2009to the ENSEMBLE gene build and further processing of the read counts was performed using Deseq Anders et al 2009 Genome build: zv9 Supplementary files format and content: The mRNA counts.csv contains the DESeq normalized read counts per Ensemble gene as indicated. Headers indicate the sample name. Zebrafish Zygotes   mRNA was extracted from zygotes or by RIP experiments followed by Trizol extraction The RNA was extracted using chloroform and precipitated with iso propanol. Ovation RNA seq System V2 NuGEN library preperation kit was used and the library was sequenced on an Illumina HISEQ using 50bp single end sequencing. Zebrafish were maintained under standard conditions. fraction:Input GSM2087139 GSM2087139: Input 1; Danio rerio; RIP Seq GSM2087139   1 mRNA was extracted from zygotes or by RIP experiments followed by Trizol extraction The RNA was extracted using chloroform and precipitated with iso propanol. Ovation RNA seq System V2 NuGEN library preperation kit was used and the library was sequenced on an Illumina HISEQ using 50bp single end sequencing. GEO Accession:GSM2087139 RIP-Seq TRANSCRIPTOMIC other SINGLE ILLUMINA Illumina HiSeq 2000   SRP071849     WT_Input_1.fastq.gz fastq 1109818905.0 21761155.0 GSM2087139 r1                       SRX1637109 SRS1342928 SRA385813 GEO Rene Ketting, RNA silencing, IMB 1 0.87467   0.18794   0.79411   0.63378   51   B   usable mapping rate illumina hiseq_era unknown other unknown sc_generic single_cell_generic generic-scrnaseq-only   Germany 2016-03-13 Zygote Embryo Oocyte Reproductive System
40704 40704 SRR3231360 SRX1637108 SRS1342929 SRP071849 PRJNA315400 Tdrd6a recruits Ziwi bound piRNAs and coordinates deposition of germ plasm mRNAs into primordial germ cells [RIP Seq] GSE79161 Transcriptome Analysis Germ plasm the Balbiani body and nuage are evolutionary conserved structures essential for germ cell specification and maintenance. We describe Tdrd6a as a component of these structures with two distinct molecular functions. First Tdrd6a facilitates the accumulation of the typical antisense bias of piRNAs without xxx effects on piRNA biogenesis signatures. Second we show that Tdrd6a is required for Balbiani body and germ plasm integrity and associates with RNA binding proteins and germ plasm mRNAs. On the cell biological level maternally contributed Tdrd6a strongly impacts germ cell formation but is dispensable for fertility. Using single cell RNA sequencing we demonstrate that Tdrd6a promotes early germ cell development and regulates the stoichiometry of germ plasm mRNAs. We propose that Tdrd6a functions as a scaffold to recruit correct ratios of germ plasm transcripts and to accumulate antisense piRNA complexes in order to ensure both specification and maintenance of germ cells. Overall design: RNA was extracted from ovary tissue or from RIP experiments by Trizol extraction. parent bioproject:PRJNA315403 pubmed:30086300   Tdrd6a IP 3 GSM2087138   tissue:Zebrafish Zygotes|fraction:Tdrd6a IP Tdrd6a IP 3 Reads were mapped to Zv9 using tophat trapnell et al 2009to the ENSEMBLE gene build and further processing of the read counts was performed using Deseq Anders et al 2009 Genome build: zv9 Supplementary files format and content: The mRNA counts.csv contains the DESeq normalized read counts per Ensemble gene as indicated. Headers indicate the sample name. Zebrafish Zygotes   mRNA was extracted from zygotes or by RIP experiments followed by Trizol extraction The RNA was extracted using chloroform and precipitated with iso propanol. Ovation RNA seq System V2 NuGEN library preperation kit was used and the library was sequenced on an Illumina HISEQ using 50bp single end sequencing. Zebrafish were maintained under standard conditions. fraction:Tdrd6a IP GSM2087138 GSM2087138: Tdrd6a IP 3; Danio rerio; RIP Seq GSM2087138   1 mRNA was extracted from zygotes or by RIP experiments followed by Trizol extraction The RNA was extracted using chloroform and precipitated with iso propanol. Ovation RNA seq System V2 NuGEN library preperation kit was used and the library was sequenced on an Illumina HISEQ using 50bp single end sequencing. GEO Accession:GSM2087138 RIP-Seq TRANSCRIPTOMIC other SINGLE ILLUMINA Illumina HiSeq 2000   SRP071849     WT_Tdrd6a_IP_3.fastq.gz fastq 843641439.0 16541989.0 GSM2087138 r1 0:51 A:216465581;C:196892096;G:207722567;T:222361382;N:199813 51       216465581 196892096 207722567 222361382 199813 SRX1637108 SRS1342929 SRA385813 GEO Rene Ketting, RNA silencing, IMB 1 0.84615   0.08957   0.78768   0.50612   51   B   usable mapping rate illumina hiseq_era unknown other unknown sc_generic single_cell_generic generic-scrnaseq-only   Germany 2016-03-13 Zygote Embryo Oocyte Reproductive System
40705 40705 SRR3231359 SRX1637107 SRS1342930 SRP071849 PRJNA315400 Tdrd6a recruits Ziwi bound piRNAs and coordinates deposition of germ plasm mRNAs into primordial germ cells [RIP Seq] GSE79161 Transcriptome Analysis Germ plasm the Balbiani body and nuage are evolutionary conserved structures essential for germ cell specification and maintenance. We describe Tdrd6a as a component of these structures with two distinct molecular functions. First Tdrd6a facilitates the accumulation of the typical antisense bias of piRNAs without xxx effects on piRNA biogenesis signatures. Second we show that Tdrd6a is required for Balbiani body and germ plasm integrity and associates with RNA binding proteins and germ plasm mRNAs. On the cell biological level maternally contributed Tdrd6a strongly impacts germ cell formation but is dispensable for fertility. Using single cell RNA sequencing we demonstrate that Tdrd6a promotes early germ cell development and regulates the stoichiometry of germ plasm mRNAs. We propose that Tdrd6a functions as a scaffold to recruit correct ratios of germ plasm transcripts and to accumulate antisense piRNA complexes in order to ensure both specification and maintenance of germ cells. Overall design: RNA was extracted from ovary tissue or from RIP experiments by Trizol extraction. parent bioproject:PRJNA315403 pubmed:30086300   Tdrd6a IP 2 GSM2087137   tissue:Zebrafish Zygotes|fraction:Tdrd6a IP Tdrd6a IP 2 Reads were mapped to Zv9 using tophat trapnell et al 2009to the ENSEMBLE gene build and further processing of the read counts was performed using Deseq Anders et al 2009 Genome build: zv9 Supplementary files format and content: The mRNA counts.csv contains the DESeq normalized read counts per Ensemble gene as indicated. Headers indicate the sample name. Zebrafish Zygotes   mRNA was extracted from zygotes or by RIP experiments followed by Trizol extraction The RNA was extracted using chloroform and precipitated with iso propanol. Ovation RNA seq System V2 NuGEN library preperation kit was used and the library was sequenced on an Illumina HISEQ using 50bp single end sequencing. Zebrafish were maintained under standard conditions. fraction:Tdrd6a IP GSM2087137 GSM2087137: Tdrd6a IP 2; Danio rerio; RIP Seq GSM2087137   1 mRNA was extracted from zygotes or by RIP experiments followed by Trizol extraction The RNA was extracted using chloroform and precipitated with iso propanol. Ovation RNA seq System V2 NuGEN library preperation kit was used and the library was sequenced on an Illumina HISEQ using 50bp single end sequencing. GEO Accession:GSM2087137 RIP-Seq TRANSCRIPTOMIC other SINGLE ILLUMINA Illumina HiSeq 2000   SRP071849     WT_Tdrd6a_IP_2.fastq.gz fastq 391198050.0 7670550.0 GSM2087137 r1 0:51 A:99015594;C:91525929;G:97155710;T:103371571;N:129246 51       99015594 91525929 97155710 103371571 129246 SRX1637107 SRS1342930 SRA385813 GEO Rene Ketting, RNA silencing, IMB 1 0.85778   0.12015   0.80515   0.47541   51   B   usable mapping rate illumina hiseq_era unknown other unknown sc_generic single_cell_generic generic-scrnaseq-only   Germany 2016-03-13 Zygote Embryo Oocyte Reproductive System
40706 40706 SRR3231358 SRX1637106 SRS1342931 SRP071849 PRJNA315400 Tdrd6a recruits Ziwi bound piRNAs and coordinates deposition of germ plasm mRNAs into primordial germ cells [RIP Seq] GSE79161 Transcriptome Analysis Germ plasm the Balbiani body and nuage are evolutionary conserved structures essential for germ cell specification and maintenance. We describe Tdrd6a as a component of these structures with two distinct molecular functions. First Tdrd6a facilitates the accumulation of the typical antisense bias of piRNAs without xxx effects on piRNA biogenesis signatures. Second we show that Tdrd6a is required for Balbiani body and germ plasm integrity and associates with RNA binding proteins and germ plasm mRNAs. On the cell biological level maternally contributed Tdrd6a strongly impacts germ cell formation but is dispensable for fertility. Using single cell RNA sequencing we demonstrate that Tdrd6a promotes early germ cell development and regulates the stoichiometry of germ plasm mRNAs. We propose that Tdrd6a functions as a scaffold to recruit correct ratios of germ plasm transcripts and to accumulate antisense piRNA complexes in order to ensure both specification and maintenance of germ cells. Overall design: RNA was extracted from ovary tissue or from RIP experiments by Trizol extraction. parent bioproject:PRJNA315403 pubmed:30086300   Tdrd6a IP 1 GSM2087136   tissue:Zebrafish Zygotes|fraction:Tdrd6a IP Tdrd6a IP 1 Reads were mapped to Zv9 using tophat trapnell et al 2009to the ENSEMBLE gene build and further processing of the read counts was performed using Deseq Anders et al 2009 Genome build: zv9 Supplementary files format and content: The mRNA counts.csv contains the DESeq normalized read counts per Ensemble gene as indicated. Headers indicate the sample name. Zebrafish Zygotes   mRNA was extracted from zygotes or by RIP experiments followed by Trizol extraction The RNA was extracted using chloroform and precipitated with iso propanol. Ovation RNA seq System V2 NuGEN library preperation kit was used and the library was sequenced on an Illumina HISEQ using 50bp single end sequencing. Zebrafish were maintained under standard conditions. fraction:Tdrd6a IP GSM2087136 GSM2087136: Tdrd6a IP 1; Danio rerio; RIP Seq GSM2087136   1 mRNA was extracted from zygotes or by RIP experiments followed by Trizol extraction The RNA was extracted using chloroform and precipitated with iso propanol. Ovation RNA seq System V2 NuGEN library preperation kit was used and the library was sequenced on an Illumina HISEQ using 50bp single end sequencing. GEO Accession:GSM2087136 RIP-Seq TRANSCRIPTOMIC other SINGLE ILLUMINA Illumina HiSeq 2000   SRP071849     WT_Tdrd6a_IP_1.fastq.gz fastq 1927415052.0 37792452.0 GSM2087136 r1 0:51 A:498125298;C:444860381;G:468826607;T:515142356;N:460410 51       498125298 444860381 468826607 515142356 460410 SRX1637106 SRS1342931 SRA385813 GEO Rene Ketting, RNA silencing, IMB 1 0.86194   0.09753   0.80146   0.47808   51   B   usable mapping rate illumina hiseq_era unknown other unknown sc_generic single_cell_generic generic-scrnaseq-only   Germany 2016-03-13 Zygote Embryo Oocyte Reproductive System
41560 41560 SRR5017063 SRX2345558 SRS1796155 SRP093295 PRJNA353372 N6 methyladenosine dynamics during early vertebrate embryogenesis GSE89815 Other Early vertebrate embryogenesis is characterized by extensive post transcriptional regulation during the maternal to zygotic transition. The N6 methyladenosine m6A modifications on mRNA has been shown to affect both translation and stability of transcripts. Here we investigate the m6A topology during early vertebrate embryogenesis and its association with RNA stability translation efficiency and effect on miR 430 degradation kinetics. Notably we find a strong association of m6A with cytoplasmic polyadenylation and translational efficiency prior to zygotic genome activation. Genes required for zygotic genome activation such as nanog and pou5f3 display dynamic m6A levels. post zygotic genome activation m6A is associated with improved stability and dampens the effect of miR 430 mediated degradation. Through sequence analyses we identified enrichment of motifs for RNA binding proteins involved in translational regulation and RNA degradation. We propose a role for m6A in multiple mRNA regulatory mechanisms for the first time in an in vivo system and improve our understanding of the combinatorial code behind the complex post transcriptional regulation of reprogramming during early vertebrate development. Overall design: Examination of m6A in four different developmental stages       input 1cell rep2 GSM2390016   tissue:Embryos|developmental stage:1cell embryos|strain:AB wild type input 1cell rep2 Base calling The sequencing output from each of the 4 lanes the sequencing run was trimmed 7 nucleotides in the 5’end and de multiplexed 4 samples in each lane. Reads were mapped to Zv10 using the STAR aligner Dobin et al. 2013 with options seedSearchStartLmax 15 clip3pNbases 10 clip5pNbases 10 outFilterMultimapNmax 20 outFilterMismatchNoverLmax 0.05 outFilterMatchNminOverLread 0.0 outFilterMatchNmin 15 outFilterScoreMinOverLread 0.0. We performed peak calling and detection of differentially methylated genes using ExomePeak Meng et al. 2013. Genome build: Zv10 Supplementary files format and content: Bed files with enriched regions from each developmental stage and txt file with raw and normalized read counts for each gene using the input samples Embryos   We isolated total RNA from 4 stages 1 cell/20 minutes post fertilization 2 4 and 6 hpf in batches of 200 embryos using TRIzol Invitrogen cat.no. 15596 018. We added ERCC spike in RNA TermoFisher scientific cat.no. 4456740 to the trizol. For each sequencing library two batches of total RNA from 200 embryos were merged and enriched for polyA+ RNA using Dynabeads® mRNA Purification Kit Ambion #61006. The m6A RIP experiment was carried out as previously described Ke et al. 2015 and the protocol can be found in supplementary file 1. Briefly polyA+ enriched RNA was partially fragmented by alkaline hydrolysis ethanol precipitated and SDS PAGE size selected for 20 80nt. Part of the fragmented RNA was used as input and the rest was immunoprecipitated at 4oC for 2 h using Dynabeads Protein A Life Technologies # 10008D conjugated anti m6A antibody Synaptic systems # 202003. post stringent washing the bound RNA was eluted with 0.5mg/ml N6 methyladenosine sodium salt Sigma Aldrich # M2780 ethanol precipitated and resuspended with RNase free water. The eluted RNA and input RNA were subjected to 3’pre adenylated DNA liker ligation with T4 RNA ligase2 truncated KQ NEB #M0373L at 16 oC over night. The sequencing library was constructed with bromodeoxyuridine BrdU CLIP protocol described in Weyn Vanhentenryck et al. 2014 with improved RT primers indicated in Ke et al. 2015. Embryos from the AB wild type strain were obtained from the NMBU zebrafish facility where the zebrafish were kept at 28±1°C on a 14 10 hour light dark cycle at a density of 5 10 fish/L. System water SW was prepared from particle and active charcoal filtrated tap water deionized by reverse osmosis RO and kept sterile by UV irradiation. The water was conditioned to a conductivity of 500µS/cm general hardness GH 4 5 and pH 7.5 by addition of 155g synthetic sea salt Instant Ocean Blacksburg USA 53g sodium carbonate and 15g calcium chloride Sigma Aldrich per liter RO water. Adult fish were fed with Gemma Micro 300 Skretting Stavanger Norway dry feed twice a day and live artemia Scanbur Karlslunde Denmark once a day. Health monitoring was by daily inspection use of sentinel fish sent for pathology ZIRC Eugene Oregon and water microbiology analysis NMBU Vetbio Oslo every six month. Adult fish were allowed to mate for 30 minutes in standard 1L breeding tanks Aquatic Habitats Apopka FL. Harvested embryos were kept in autoclaved SW at 28 °C harvested by snap freezing liquid nitrogen and visually controlled for stage and lack of abnormalities at the selected time points. All experiments were performed according to Norwegian Animal Welfare Act 2009 the EU Directive 2010/63. developmental stage:1cell embryos|strain:AB wild type GSM2390016 GSM2390016: input 1cell rep2; Danio rerio; RIP Seq GSM2390016   1 We isolated total RNA from 4 stages 1 cell/20 minutes post fertilization 2 4 and 6 hpf in batches of 200 embryos using TRIzol Invitrogen cat.no. 15596 018. We added ERCC spike in RNA TermoFisher scientific cat.no. 4456740 to the trizol. For each sequencing library two batches of total RNA from 200 embryos were merged and enriched for polyA+ RNA using Dynabeads® mRNA Purification Kit Ambion #61006. The m6A RIP experiment was carried out as previously described Ke et al. 2015 and the protocol can be found in supplementary file 1. Briefly polyA+ enriched RNA was partially fragmented by alkaline hydrolysis ethanol precipitated and SDS PAGE size selected for 20 80nt. Part of the fragmented RNA was used as input and the rest was immunoprecipitated at 4oC for 2 h using Dynabeads Protein A Life Technologies # 10008D conjugated anti m6A antibody Synaptic systems # 202003. post stringent washing the bound RNA was eluted with 0.5mg/ml N6 methyladenosine sodium salt Sigma Aldrich # M2780 ethanol precipitated and resuspended with RNase free water. The eluted RNA and input RNA were subjected to 3’pre adenylated DNA liker ligation with T4 RNA ligase2 truncated KQ NEB #M0373L at 16 oC over night. The sequencing library was constructed with bromodeoxyuridine BrdU CLIP protocol described in Weyn Vanhentenryck et al. 2014 with improved RT primers indicated in Ke et al. 2015. GEO Accession:GSM2390016 RIP-Seq TRANSCRIPTOMIC other SINGLE ILLUMINA Illumina HiSeq 2000   SRP093295     input_1cell_rep2.fastq.gz fastq 3379762010.0 35954915.0 GSM2390016 r1 0:94 A:898920912;C:816234808;G:823428476;T:840956466;N:221348 94       898920912 816234808 823428476 840956466 221348 SRX2345558 SRS1796155 SRA492943 GEO Klungland Lab, Dept of microbiology, Oslo University Hospital 1 0.04513   0.00864   0.95085   0.51692   94   B   usable mapping rate illumina hiseq_era unknown poly_a unknown bulk unknown unknown   Norway 2016-11-14 Zygote Embryo Embryo Imprecise All anatomical structures
41561 41561 SRR5017062 SRX2345557 SRS1796128 SRP093295 PRJNA353372 N6 methyladenosine dynamics during early vertebrate embryogenesis GSE89815 Other Early vertebrate embryogenesis is characterized by extensive post transcriptional regulation during the maternal to zygotic transition. The N6 methyladenosine m6A modifications on mRNA has been shown to affect both translation and stability of transcripts. Here we investigate the m6A topology during early vertebrate embryogenesis and its association with RNA stability translation efficiency and effect on miR 430 degradation kinetics. Notably we find a strong association of m6A with cytoplasmic polyadenylation and translational efficiency prior to zygotic genome activation. Genes required for zygotic genome activation such as nanog and pou5f3 display dynamic m6A levels. post zygotic genome activation m6A is associated with improved stability and dampens the effect of miR 430 mediated degradation. Through sequence analyses we identified enrichment of motifs for RNA binding proteins involved in translational regulation and RNA degradation. We propose a role for m6A in multiple mRNA regulatory mechanisms for the first time in an in vivo system and improve our understanding of the combinatorial code behind the complex post transcriptional regulation of reprogramming during early vertebrate development. Overall design: Examination of m6A in four different developmental stages       input 1cell rep1 GSM2390015   tissue:Embryos|developmental stage:1cell embryos|strain:AB wild type input 1cell rep1 Base calling The sequencing output from each of the 4 lanes the sequencing run was trimmed 7 nucleotides in the 5’end and de multiplexed 4 samples in each lane. Reads were mapped to Zv10 using the STAR aligner Dobin et al. 2013 with options seedSearchStartLmax 15 clip3pNbases 10 clip5pNbases 10 outFilterMultimapNmax 20 outFilterMismatchNoverLmax 0.05 outFilterMatchNminOverLread 0.0 outFilterMatchNmin 15 outFilterScoreMinOverLread 0.0. We performed peak calling and detection of differentially methylated genes using ExomePeak Meng et al. 2013. Genome build: Zv10 Supplementary files format and content: Bed files with enriched regions from each developmental stage and txt file with raw and normalized read counts for each gene using the input samples Embryos   We isolated total RNA from 4 stages 1 cell/20 minutes post fertilization 2 4 and 6 hpf in batches of 200 embryos using TRIzol Invitrogen cat.no. 15596 018. We added ERCC spike in RNA TermoFisher scientific cat.no. 4456740 to the trizol. For each sequencing library two batches of total RNA from 200 embryos were merged and enriched for polyA+ RNA using Dynabeads® mRNA Purification Kit Ambion #61006. The m6A RIP experiment was carried out as previously described Ke et al. 2015 and the protocol can be found in supplementary file 1. Briefly polyA+ enriched RNA was partially fragmented by alkaline hydrolysis ethanol precipitated and SDS PAGE size selected for 20 80nt. Part of the fragmented RNA was used as input and the rest was immunoprecipitated at 4oC for 2 h using Dynabeads Protein A Life Technologies # 10008D conjugated anti m6A antibody Synaptic systems # 202003. post stringent washing the bound RNA was eluted with 0.5mg/ml N6 methyladenosine sodium salt Sigma Aldrich # M2780 ethanol precipitated and resuspended with RNase free water. The eluted RNA and input RNA were subjected to 3’pre adenylated DNA liker ligation with T4 RNA ligase2 truncated KQ NEB #M0373L at 16 oC over night. The sequencing library was constructed with bromodeoxyuridine BrdU CLIP protocol described in Weyn Vanhentenryck et al. 2014 with improved RT primers indicated in Ke et al. 2015. Embryos from the AB wild type strain were obtained from the NMBU zebrafish facility where the zebrafish were kept at 28±1°C on a 14 10 hour light dark cycle at a density of 5 10 fish/L. System water SW was prepared from particle and active charcoal filtrated tap water deionized by reverse osmosis RO and kept sterile by UV irradiation. The water was conditioned to a conductivity of 500µS/cm general hardness GH 4 5 and pH 7.5 by addition of 155g synthetic sea salt Instant Ocean Blacksburg USA 53g sodium carbonate and 15g calcium chloride Sigma Aldrich per liter RO water. Adult fish were fed with Gemma Micro 300 Skretting Stavanger Norway dry feed twice a day and live artemia Scanbur Karlslunde Denmark once a day. Health monitoring was by daily inspection use of sentinel fish sent for pathology ZIRC Eugene Oregon and water microbiology analysis NMBU Vetbio Oslo every six month. Adult fish were allowed to mate for 30 minutes in standard 1L breeding tanks Aquatic Habitats Apopka FL. Harvested embryos were kept in autoclaved SW at 28 °C harvested by snap freezing liquid nitrogen and visually controlled for stage and lack of abnormalities at the selected time points. All experiments were performed according to Norwegian Animal Welfare Act 2009 the EU Directive 2010/63. developmental stage:1cell embryos|strain:AB wild type GSM2390015 GSM2390015: input 1cell rep1; Danio rerio; RIP Seq GSM2390015   1 We isolated total RNA from 4 stages 1 cell/20 minutes post fertilization 2 4 and 6 hpf in batches of 200 embryos using TRIzol Invitrogen cat.no. 15596 018. We added ERCC spike in RNA TermoFisher scientific cat.no. 4456740 to the trizol. For each sequencing library two batches of total RNA from 200 embryos were merged and enriched for polyA+ RNA using Dynabeads® mRNA Purification Kit Ambion #61006. The m6A RIP experiment was carried out as previously described Ke et al. 2015 and the protocol can be found in supplementary file 1. Briefly polyA+ enriched RNA was partially fragmented by alkaline hydrolysis ethanol precipitated and SDS PAGE size selected for 20 80nt. Part of the fragmented RNA was used as input and the rest was immunoprecipitated at 4oC for 2 h using Dynabeads Protein A Life Technologies # 10008D conjugated anti m6A antibody Synaptic systems # 202003. post stringent washing the bound RNA was eluted with 0.5mg/ml N6 methyladenosine sodium salt Sigma Aldrich # M2780 ethanol precipitated and resuspended with RNase free water. The eluted RNA and input RNA were subjected to 3’pre adenylated DNA liker ligation with T4 RNA ligase2 truncated KQ NEB #M0373L at 16 oC over night. The sequencing library was constructed with bromodeoxyuridine BrdU CLIP protocol described in Weyn Vanhentenryck et al. 2014 with improved RT primers indicated in Ke et al. 2015. GEO Accession:GSM2390015 RIP-Seq TRANSCRIPTOMIC other SINGLE ILLUMINA Illumina HiSeq 2000   SRP093295     input_1cell_rep1.fastq.gz fastq 3650321646.0 38833209.0 GSM2390015 r1 0:94 A:951411355;C:878458528;G:890250453;T:929968044;N:233266 94       951411355 878458528 890250453 929968044 233266 SRX2345557 SRS1796128 SRA492943 GEO Klungland Lab, Dept of microbiology, Oslo University Hospital 1 0.03728   0.00668   0.94901   0.60038   94   B   usable mapping rate illumina hiseq_era unknown poly_a unknown bulk unknown unknown   Norway 2016-11-14 Zygote Embryo Embryo Imprecise All anatomical structures
41562 41562 SRR5017061 SRX2345556 SRS1796127 SRP093295 PRJNA353372 N6 methyladenosine dynamics during early vertebrate embryogenesis GSE89815 Other Early vertebrate embryogenesis is characterized by extensive post transcriptional regulation during the maternal to zygotic transition. The N6 methyladenosine m6A modifications on mRNA has been shown to affect both translation and stability of transcripts. Here we investigate the m6A topology during early vertebrate embryogenesis and its association with RNA stability translation efficiency and effect on miR 430 degradation kinetics. Notably we find a strong association of m6A with cytoplasmic polyadenylation and translational efficiency prior to zygotic genome activation. Genes required for zygotic genome activation such as nanog and pou5f3 display dynamic m6A levels. post zygotic genome activation m6A is associated with improved stability and dampens the effect of miR 430 mediated degradation. Through sequence analyses we identified enrichment of motifs for RNA binding proteins involved in translational regulation and RNA degradation. We propose a role for m6A in multiple mRNA regulatory mechanisms for the first time in an in vivo system and improve our understanding of the combinatorial code behind the complex post transcriptional regulation of reprogramming during early vertebrate development. Overall design: Examination of m6A in four different developmental stages       ip 1cell rep2 GSM2390014   tissue:Embryos|developmental stage:1cell embryos|strain:AB wild type ip 1cell rep2 Base calling The sequencing output from each of the 4 lanes the sequencing run was trimmed 7 nucleotides in the 5’end and de multiplexed 4 samples in each lane. Reads were mapped to Zv10 using the STAR aligner Dobin et al. 2013 with options seedSearchStartLmax 15 clip3pNbases 10 clip5pNbases 10 outFilterMultimapNmax 20 outFilterMismatchNoverLmax 0.05 outFilterMatchNminOverLread 0.0 outFilterMatchNmin 15 outFilterScoreMinOverLread 0.0. We performed peak calling and detection of differentially methylated genes using ExomePeak Meng et al. 2013. Genome build: Zv10 Supplementary files format and content: Bed files with enriched regions from each developmental stage and txt file with raw and normalized read counts for each gene using the input samples Embryos   We isolated total RNA from 4 stages 1 cell/20 minutes post fertilization 2 4 and 6 hpf in batches of 200 embryos using TRIzol Invitrogen cat.no. 15596 018. We added ERCC spike in RNA TermoFisher scientific cat.no. 4456740 to the trizol. For each sequencing library two batches of total RNA from 200 embryos were merged and enriched for polyA+ RNA using Dynabeads® mRNA Purification Kit Ambion #61006. The m6A RIP experiment was carried out as previously described Ke et al. 2015 and the protocol can be found in supplementary file 1. Briefly polyA+ enriched RNA was partially fragmented by alkaline hydrolysis ethanol precipitated and SDS PAGE size selected for 20 80nt. Part of the fragmented RNA was used as input and the rest was immunoprecipitated at 4oC for 2 h using Dynabeads Protein A Life Technologies # 10008D conjugated anti m6A antibody Synaptic systems # 202003. post stringent washing the bound RNA was eluted with 0.5mg/ml N6 methyladenosine sodium salt Sigma Aldrich # M2780 ethanol precipitated and resuspended with RNase free water. The eluted RNA and input RNA were subjected to 3’pre adenylated DNA liker ligation with T4 RNA ligase2 truncated KQ NEB #M0373L at 16 oC over night. The sequencing library was constructed with bromodeoxyuridine BrdU CLIP protocol described in Weyn Vanhentenryck et al. 2014 with improved RT primers indicated in Ke et al. 2015. Embryos from the AB wild type strain were obtained from the NMBU zebrafish facility where the zebrafish were kept at 28±1°C on a 14 10 hour light dark cycle at a density of 5 10 fish/L. System water SW was prepared from particle and active charcoal filtrated tap water deionized by reverse osmosis RO and kept sterile by UV irradiation. The water was conditioned to a conductivity of 500µS/cm general hardness GH 4 5 and pH 7.5 by addition of 155g synthetic sea salt Instant Ocean Blacksburg USA 53g sodium carbonate and 15g calcium chloride Sigma Aldrich per liter RO water. Adult fish were fed with Gemma Micro 300 Skretting Stavanger Norway dry feed twice a day and live artemia Scanbur Karlslunde Denmark once a day. Health monitoring was by daily inspection use of sentinel fish sent for pathology ZIRC Eugene Oregon and water microbiology analysis NMBU Vetbio Oslo every six month. Adult fish were allowed to mate for 30 minutes in standard 1L breeding tanks Aquatic Habitats Apopka FL. Harvested embryos were kept in autoclaved SW at 28 °C harvested by snap freezing liquid nitrogen and visually controlled for stage and lack of abnormalities at the selected time points. All experiments were performed according to Norwegian Animal Welfare Act 2009 the EU Directive 2010/63. developmental stage:1cell embryos|strain:AB wild type GSM2390014 GSM2390014: ip 1cell rep2; Danio rerio; RIP Seq GSM2390014   1 We isolated total RNA from 4 stages 1 cell/20 minutes post fertilization 2 4 and 6 hpf in batches of 200 embryos using TRIzol Invitrogen cat.no. 15596 018. We added ERCC spike in RNA TermoFisher scientific cat.no. 4456740 to the trizol. For each sequencing library two batches of total RNA from 200 embryos were merged and enriched for polyA+ RNA using Dynabeads® mRNA Purification Kit Ambion #61006. The m6A RIP experiment was carried out as previously described Ke et al. 2015 and the protocol can be found in supplementary file 1. Briefly polyA+ enriched RNA was partially fragmented by alkaline hydrolysis ethanol precipitated and SDS PAGE size selected for 20 80nt. Part of the fragmented RNA was used as input and the rest was immunoprecipitated at 4oC for 2 h using Dynabeads Protein A Life Technologies # 10008D conjugated anti m6A antibody Synaptic systems # 202003. post stringent washing the bound RNA was eluted with 0.5mg/ml N6 methyladenosine sodium salt Sigma Aldrich # M2780 ethanol precipitated and resuspended with RNase free water. The eluted RNA and input RNA were subjected to 3’pre adenylated DNA liker ligation with T4 RNA ligase2 truncated KQ NEB #M0373L at 16 oC over night. The sequencing library was constructed with bromodeoxyuridine BrdU CLIP protocol described in Weyn Vanhentenryck et al. 2014 with improved RT primers indicated in Ke et al. 2015. GEO Accession:GSM2390014 RIP-Seq TRANSCRIPTOMIC other SINGLE ILLUMINA Illumina HiSeq 2000   SRP093295     ip_1cell_rep2.fastq.gz fastq 3485432580.0 37079070.0 GSM2390014 r1 0:94 A:953699047;C:835179152;G:846963064;T:848487912;N:1103405 94       953699047 835179152 846963064 848487912 1103405 SRX2345556 SRS1796127 SRA492943 GEO Klungland Lab, Dept of microbiology, Oslo University Hospital 1 0.02534   0.00219   0.95891   0.5975   94   B   usable mapping rate illumina hiseq_era unknown poly_a unknown bulk unknown unknown   Norway 2016-11-14 Zygote Embryo Embryo Imprecise All anatomical structures
41563 41563 SRR5017060 SRX2345555 SRS1796130 SRP093295 PRJNA353372 N6 methyladenosine dynamics during early vertebrate embryogenesis GSE89815 Other Early vertebrate embryogenesis is characterized by extensive post transcriptional regulation during the maternal to zygotic transition. The N6 methyladenosine m6A modifications on mRNA has been shown to affect both translation and stability of transcripts. Here we investigate the m6A topology during early vertebrate embryogenesis and its association with RNA stability translation efficiency and effect on miR 430 degradation kinetics. Notably we find a strong association of m6A with cytoplasmic polyadenylation and translational efficiency prior to zygotic genome activation. Genes required for zygotic genome activation such as nanog and pou5f3 display dynamic m6A levels. post zygotic genome activation m6A is associated with improved stability and dampens the effect of miR 430 mediated degradation. Through sequence analyses we identified enrichment of motifs for RNA binding proteins involved in translational regulation and RNA degradation. We propose a role for m6A in multiple mRNA regulatory mechanisms for the first time in an in vivo system and improve our understanding of the combinatorial code behind the complex post transcriptional regulation of reprogramming during early vertebrate development. Overall design: Examination of m6A in four different developmental stages       ip 1cell rep1 GSM2390013   tissue:Embryos|developmental stage:1cell embryos|strain:AB wild type ip 1cell rep1 Base calling The sequencing output from each of the 4 lanes the sequencing run was trimmed 7 nucleotides in the 5’end and de multiplexed 4 samples in each lane. Reads were mapped to Zv10 using the STAR aligner Dobin et al. 2013 with options seedSearchStartLmax 15 clip3pNbases 10 clip5pNbases 10 outFilterMultimapNmax 20 outFilterMismatchNoverLmax 0.05 outFilterMatchNminOverLread 0.0 outFilterMatchNmin 15 outFilterScoreMinOverLread 0.0. We performed peak calling and detection of differentially methylated genes using ExomePeak Meng et al. 2013. Genome build: Zv10 Supplementary files format and content: Bed files with enriched regions from each developmental stage and txt file with raw and normalized read counts for each gene using the input samples Embryos   We isolated total RNA from 4 stages 1 cell/20 minutes post fertilization 2 4 and 6 hpf in batches of 200 embryos using TRIzol Invitrogen cat.no. 15596 018. We added ERCC spike in RNA TermoFisher scientific cat.no. 4456740 to the trizol. For each sequencing library two batches of total RNA from 200 embryos were merged and enriched for polyA+ RNA using Dynabeads® mRNA Purification Kit Ambion #61006. The m6A RIP experiment was carried out as previously described Ke et al. 2015 and the protocol can be found in supplementary file 1. Briefly polyA+ enriched RNA was partially fragmented by alkaline hydrolysis ethanol precipitated and SDS PAGE size selected for 20 80nt. Part of the fragmented RNA was used as input and the rest was immunoprecipitated at 4oC for 2 h using Dynabeads Protein A Life Technologies # 10008D conjugated anti m6A antibody Synaptic systems # 202003. post stringent washing the bound RNA was eluted with 0.5mg/ml N6 methyladenosine sodium salt Sigma Aldrich # M2780 ethanol precipitated and resuspended with RNase free water. The eluted RNA and input RNA were subjected to 3’pre adenylated DNA liker ligation with T4 RNA ligase2 truncated KQ NEB #M0373L at 16 oC over night. The sequencing library was constructed with bromodeoxyuridine BrdU CLIP protocol described in Weyn Vanhentenryck et al. 2014 with improved RT primers indicated in Ke et al. 2015. Embryos from the AB wild type strain were obtained from the NMBU zebrafish facility where the zebrafish were kept at 28±1°C on a 14 10 hour light dark cycle at a density of 5 10 fish/L. System water SW was prepared from particle and active charcoal filtrated tap water deionized by reverse osmosis RO and kept sterile by UV irradiation. The water was conditioned to a conductivity of 500µS/cm general hardness GH 4 5 and pH 7.5 by addition of 155g synthetic sea salt Instant Ocean Blacksburg USA 53g sodium carbonate and 15g calcium chloride Sigma Aldrich per liter RO water. Adult fish were fed with Gemma Micro 300 Skretting Stavanger Norway dry feed twice a day and live artemia Scanbur Karlslunde Denmark once a day. Health monitoring was by daily inspection use of sentinel fish sent for pathology ZIRC Eugene Oregon and water microbiology analysis NMBU Vetbio Oslo every six month. Adult fish were allowed to mate for 30 minutes in standard 1L breeding tanks Aquatic Habitats Apopka FL. Harvested embryos were kept in autoclaved SW at 28 °C harvested by snap freezing liquid nitrogen and visually controlled for stage and lack of abnormalities at the selected time points. All experiments were performed according to Norwegian Animal Welfare Act 2009 the EU Directive 2010/63. developmental stage:1cell embryos|strain:AB wild type GSM2390013 GSM2390013: ip 1cell rep1; Danio rerio; RIP Seq GSM2390013   1 We isolated total RNA from 4 stages 1 cell/20 minutes post fertilization 2 4 and 6 hpf in batches of 200 embryos using TRIzol Invitrogen cat.no. 15596 018. We added ERCC spike in RNA TermoFisher scientific cat.no. 4456740 to the trizol. For each sequencing library two batches of total RNA from 200 embryos were merged and enriched for polyA+ RNA using Dynabeads® mRNA Purification Kit Ambion #61006. The m6A RIP experiment was carried out as previously described Ke et al. 2015 and the protocol can be found in supplementary file 1. Briefly polyA+ enriched RNA was partially fragmented by alkaline hydrolysis ethanol precipitated and SDS PAGE size selected for 20 80nt. Part of the fragmented RNA was used as input and the rest was immunoprecipitated at 4oC for 2 h using Dynabeads Protein A Life Technologies # 10008D conjugated anti m6A antibody Synaptic systems # 202003. post stringent washing the bound RNA was eluted with 0.5mg/ml N6 methyladenosine sodium salt Sigma Aldrich # M2780 ethanol precipitated and resuspended with RNase free water. The eluted RNA and input RNA were subjected to 3’pre adenylated DNA liker ligation with T4 RNA ligase2 truncated KQ NEB #M0373L at 16 oC over night. The sequencing library was constructed with bromodeoxyuridine BrdU CLIP protocol described in Weyn Vanhentenryck et al. 2014 with improved RT primers indicated in Ke et al. 2015. GEO Accession:GSM2390013 RIP-Seq TRANSCRIPTOMIC other SINGLE ILLUMINA Illumina HiSeq 2000   SRP093295     ip_1cell_rep1.fastq.gz fastq 3610167102.0 38406033.0 GSM2390013 r1 0:94 A:961438376;C:875781200;G:891055201;T:880749228;N:1143097 94       961438376 875781200 891055201 880749228 1143097 SRX2345555 SRS1796130 SRA492943 GEO Klungland Lab, Dept of microbiology, Oslo University Hospital 1 0.01939   0.00159   0.96457   0.53652   94   B   usable mapping rate illumina hiseq_era unknown poly_a unknown bulk unknown unknown   Norway 2016-11-14 Zygote Embryo Embryo Imprecise All anatomical structures
49541 49541 SRR8937003 SRX5717516 SRS4655936 SRP162876 PRJNA493828 Ybx1 binding sites and Ybx1 binding m5C sites in zebrafish embryo sample. GSE120646 Other The maternal to zygotic transition MZT is a conserved and fundamental process during which the embryo undergoes dramatic reprogramming to convert maternal environment to embryonic driven programing. However how the maternally supplied transcripts are dynamically regulated during MZT remains largely unknown. Herein through genome wide profiling of RNA 5 methylcytosine m5C in zebrafish early embryos we show that m5C methylated maternal mRNAs display higher stability during MZT. We identify that the Y box binding protein 1 Ybx1 prefers to recognizing m5C modified mRNAs through p p interaction with a key residue Trp45 in its cold shock domain CSD which plays essential roles in maternal mRNA stability and early embryogenesis of zebrafish. Cooperated with an mRNA stabilizer Pabpc1a Ybx1 promotes the stability of its target mRNAs in an m5C dependent manner. Our study demonstrates a novel mechanism of RNA m5C methylation regulated maternal mRNA stability during zebrafish MZT highlighting the critical role of m5C mRNA methylation in early development. Overall design: Examination of Ybx1 binding sites and Ybx1 binding m5C sites in zebrafish embryo. RIP seq: Briefly 500 zebrafish embryos at shield stage 4 hpf were resuspended with 2 ml lysis buffer 150 mM KCl 10 mM HEPES pH 7.6 2 mM EDTA 0.5% NP 40 0.5 mM DTT 1:100 protease inhibitor cocktail 0.4 U/μl RNasin by rotating at 4°C for 30 min and centrifuged at 15 000 g for 15 min. The supernatant was collected and pre cleared with protein A Dynabeads by rotation at 4°C for 1 h. 50 μl pre cleared embryo lysate was saved as Input and the left were incubated with affinity purified rabbit polyclonal anti Ybx1 antibody prepared by AbMax China and 30 μl Protein A Dynabeads for 4 h at 4°C or overnight. post washing with1 ml ice cold NT2 buffer 200 mM NaCl 50 mM HEPES pH 7.6 2 mM EDTA 0.05% NP 40 0.5 mM DTT 0.4U/µl RNasin for eight times and once with 1ml ice cold 1× PK buffer 100 mM Tris HCl pH 7.4 50 mM NaCl 10 mM EDTA 0.2% SDS the beads was treated i… parent bioproject:PRJNA534030     RIP INPUT 0hpf rep2 GSM3732423   source name:RIP INPUT 0hpf|strain:AB strain|age:0 hpf|tissue:whole embryo RIP INPUT 0hpf rep2 Reads were aligned to the zv9 genome assembly using TopHat v2.1.1. MACS2 v2.1.1 were used for the peak calling and peaks were annotated by applying BEDTools’ intersectBed. Genome build: zv9 Supplementary files format and content: Ybx1 binding sites in two biological replicates. RIP INPUT 0hpf   Total RNA was isolated from zebrafish embryos harvested at different stages using TRIzol® Reagent Ambion. mRNA was extracted with using Dynabeads® mRNA Purification Kit Ambion and subjected to TURBO™ DNase Invitrogen treatment at 37°C for 30 min and ethanol precipitation. Thus purified mRNA was used for RNA BisSeq library construction. RNA was extracted and subjected to library construction using Smarter smRNA Seq kit Takara. All the libraries were sequenced using HiSeq2500 Illumina in paired read mode creating reads with a length of 150 bp.   strain:AB strain|age:0 hpf|tissue:whole embryo GSM3732423 GSM3732423: RIP INPUT 0hpf rep2; Danio rerio; RIP Seq GSM3732423   1 Total RNA was isolated from zebrafish embryos harvested at different stages using TRIzol® Reagent Ambion. mRNA was extracted with using Dynabeads® mRNA Purification Kit Ambion and subjected to TURBO™ DNase Invitrogen treatment at 37°C for 30 min and ethanol precipitation. Thus purified mRNA was used for RNA BisSeq library construction. RNA was extracted and subjected to library construction using Smarter smRNA Seq kit Takara. All the libraries were sequenced using HiSeq2500 Illumina in paired read mode creating reads with a length of 150 bp. GEO Accession:GSM3732423 RIP-Seq TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina HiSeq 2500   SRP162876     RIP-INPUT_0hpf_rep2_R1.fastq.gz RIP-INPUT_0hpf_rep2_R2.fastq.gz fastq fastq 17402218800.0 58007396.0 GSM3732423 r1 0:150 1:150 A:3416144403;C:5315174821;G:5345201218;T:3320906221;N:4792137 150 150     3416144403 5315174821 5345201218 3320906221 4792137 SRX5717516 SRS4655936 SRA786939 GEO Beijing Institute of Genomics (BIG) of Chinese Academy of Sciences (CAS) 2 0.9819 0.9814 0.33605 0.34016 0.93458 0.93963 0.90172 0.91272 150 150 B B biological fallback assumption illumina hiseq_era full_length random_priming smarter bulk clip iclip   China 2019-04-22 Zygote Embryo Whole Organism All anatomical structures
49542 49542 SRR8937002 SRX5717515 SRS4655935 SRP162876 PRJNA493828 Ybx1 binding sites and Ybx1 binding m5C sites in zebrafish embryo sample. GSE120646 Other The maternal to zygotic transition MZT is a conserved and fundamental process during which the embryo undergoes dramatic reprogramming to convert maternal environment to embryonic driven programing. However how the maternally supplied transcripts are dynamically regulated during MZT remains largely unknown. Herein through genome wide profiling of RNA 5 methylcytosine m5C in zebrafish early embryos we show that m5C methylated maternal mRNAs display higher stability during MZT. We identify that the Y box binding protein 1 Ybx1 prefers to recognizing m5C modified mRNAs through p p interaction with a key residue Trp45 in its cold shock domain CSD which plays essential roles in maternal mRNA stability and early embryogenesis of zebrafish. Cooperated with an mRNA stabilizer Pabpc1a Ybx1 promotes the stability of its target mRNAs in an m5C dependent manner. Our study demonstrates a novel mechanism of RNA m5C methylation regulated maternal mRNA stability during zebrafish MZT highlighting the critical role of m5C mRNA methylation in early development. Overall design: Examination of Ybx1 binding sites and Ybx1 binding m5C sites in zebrafish embryo. RIP seq: Briefly 500 zebrafish embryos at shield stage 4 hpf were resuspended with 2 ml lysis buffer 150 mM KCl 10 mM HEPES pH 7.6 2 mM EDTA 0.5% NP 40 0.5 mM DTT 1:100 protease inhibitor cocktail 0.4 U/μl RNasin by rotating at 4°C for 30 min and centrifuged at 15 000 g for 15 min. The supernatant was collected and pre cleared with protein A Dynabeads by rotation at 4°C for 1 h. 50 μl pre cleared embryo lysate was saved as Input and the left were incubated with affinity purified rabbit polyclonal anti Ybx1 antibody prepared by AbMax China and 30 μl Protein A Dynabeads for 4 h at 4°C or overnight. post washing with1 ml ice cold NT2 buffer 200 mM NaCl 50 mM HEPES pH 7.6 2 mM EDTA 0.05% NP 40 0.5 mM DTT 0.4U/µl RNasin for eight times and once with 1ml ice cold 1× PK buffer 100 mM Tris HCl pH 7.4 50 mM NaCl 10 mM EDTA 0.2% SDS the beads was treated i… parent bioproject:PRJNA534030     RIP INPUT 0hpf rep1 GSM3732422   source name:RIP INPUT 0hpf|strain:AB strain|age:0 hpf|tissue:whole embryo RIP INPUT 0hpf rep1 Reads were aligned to the zv9 genome assembly using TopHat v2.1.1. MACS2 v2.1.1 were used for the peak calling and peaks were annotated by applying BEDTools’ intersectBed. Genome build: zv9 Supplementary files format and content: Ybx1 binding sites in two biological replicates. RIP INPUT 0hpf   Total RNA was isolated from zebrafish embryos harvested at different stages using TRIzol® Reagent Ambion. mRNA was extracted with using Dynabeads® mRNA Purification Kit Ambion and subjected to TURBO™ DNase Invitrogen treatment at 37°C for 30 min and ethanol precipitation. Thus purified mRNA was used for RNA BisSeq library construction. RNA was extracted and subjected to library construction using Smarter smRNA Seq kit Takara. All the libraries were sequenced using HiSeq2500 Illumina in paired read mode creating reads with a length of 150 bp.   strain:AB strain|age:0 hpf|tissue:whole embryo GSM3732422 GSM3732422: RIP INPUT 0hpf rep1; Danio rerio; RIP Seq GSM3732422   1 Total RNA was isolated from zebrafish embryos harvested at different stages using TRIzol® Reagent Ambion. mRNA was extracted with using Dynabeads® mRNA Purification Kit Ambion and subjected to TURBO™ DNase Invitrogen treatment at 37°C for 30 min and ethanol precipitation. Thus purified mRNA was used for RNA BisSeq library construction. RNA was extracted and subjected to library construction using Smarter smRNA Seq kit Takara. All the libraries were sequenced using HiSeq2500 Illumina in paired read mode creating reads with a length of 150 bp. GEO Accession:GSM3732422 RIP-Seq TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina HiSeq 2500   SRP162876     RIP-INPUT_0hpf_rep1_R1.fastq.gz RIP-INPUT_0hpf_rep1_R2.fastq.gz fastq fastq 16615204800.0 55384016.0 GSM3732422 r1 0:150 1:150 A:2869477028;C:5392959051;G:5575878422;T:2776252538;N:637761 150 150     2869477028 5392959051 5575878422 2776252538 637761 SRX5717515 SRS4655935 SRA786939 GEO Beijing Institute of Genomics (BIG) of Chinese Academy of Sciences (CAS) 2 0.97584 0.96561 0.04007 0.03832 0.9262 0.933 0.94642 0.94376 150 150 B B biological fallback assumption illumina hiseq_era full_length random_priming smarter bulk clip iclip   China 2019-04-22 Zygote Embryo Whole Organism All anatomical structures
49545 49545 SRR8936999 SRX5717512 SRS4655932 SRP162876 PRJNA493828 Ybx1 binding sites and Ybx1 binding m5C sites in zebrafish embryo sample. GSE120646 Other The maternal to zygotic transition MZT is a conserved and fundamental process during which the embryo undergoes dramatic reprogramming to convert maternal environment to embryonic driven programing. However how the maternally supplied transcripts are dynamically regulated during MZT remains largely unknown. Herein through genome wide profiling of RNA 5 methylcytosine m5C in zebrafish early embryos we show that m5C methylated maternal mRNAs display higher stability during MZT. We identify that the Y box binding protein 1 Ybx1 prefers to recognizing m5C modified mRNAs through p p interaction with a key residue Trp45 in its cold shock domain CSD which plays essential roles in maternal mRNA stability and early embryogenesis of zebrafish. Cooperated with an mRNA stabilizer Pabpc1a Ybx1 promotes the stability of its target mRNAs in an m5C dependent manner. Our study demonstrates a novel mechanism of RNA m5C methylation regulated maternal mRNA stability during zebrafish MZT highlighting the critical role of m5C mRNA methylation in early development. Overall design: Examination of Ybx1 binding sites and Ybx1 binding m5C sites in zebrafish embryo. RIP seq: Briefly 500 zebrafish embryos at shield stage 4 hpf were resuspended with 2 ml lysis buffer 150 mM KCl 10 mM HEPES pH 7.6 2 mM EDTA 0.5% NP 40 0.5 mM DTT 1:100 protease inhibitor cocktail 0.4 U/μl RNasin by rotating at 4°C for 30 min and centrifuged at 15 000 g for 15 min. The supernatant was collected and pre cleared with protein A Dynabeads by rotation at 4°C for 1 h. 50 μl pre cleared embryo lysate was saved as Input and the left were incubated with affinity purified rabbit polyclonal anti Ybx1 antibody prepared by AbMax China and 30 μl Protein A Dynabeads for 4 h at 4°C or overnight. post washing with1 ml ice cold NT2 buffer 200 mM NaCl 50 mM HEPES pH 7.6 2 mM EDTA 0.05% NP 40 0.5 mM DTT 0.4U/µl RNasin for eight times and once with 1ml ice cold 1× PK buffer 100 mM Tris HCl pH 7.4 50 mM NaCl 10 mM EDTA 0.2% SDS the beads was treated i… parent bioproject:PRJNA534030     RIP 0hpf rep2 GSM3732419   source name:RIP 0hpf|strain:AB strain|age:0 hpf|tissue:whole embryo|rip antibody:rabbit polyclonal anti Ybx1 antibody RIP 0hpf rep2 Reads were aligned to the zv9 genome assembly using TopHat v2.1.1. MACS2 v2.1.1 were used for the peak calling and peaks were annotated by applying BEDTools’ intersectBed. Genome build: zv9 Supplementary files format and content: Ybx1 binding sites in two biological replicates. RIP 0hpf   Total RNA was isolated from zebrafish embryos harvested at different stages using TRIzol® Reagent Ambion. mRNA was extracted with using Dynabeads® mRNA Purification Kit Ambion and subjected to TURBO™ DNase Invitrogen treatment at 37°C for 30 min and ethanol precipitation. Thus purified mRNA was used for RNA BisSeq library construction. RNA was extracted and subjected to library construction using Smarter smRNA Seq kit Takara. All the libraries were sequenced using HiSeq2500 Illumina in paired read mode creating reads with a length of 150 bp.   strain:AB strain|age:0 hpf|tissue:whole embryo|rip antibody:rabbit polyclonal anti Ybx1 antibody GSM3732419 GSM3732419: RIP 0hpf rep2; Danio rerio; RIP Seq GSM3732419   1 Total RNA was isolated from zebrafish embryos harvested at different stages using TRIzol® Reagent Ambion. mRNA was extracted with using Dynabeads® mRNA Purification Kit Ambion and subjected to TURBO™ DNase Invitrogen treatment at 37°C for 30 min and ethanol precipitation. Thus purified mRNA was used for RNA BisSeq library construction. RNA was extracted and subjected to library construction using Smarter smRNA Seq kit Takara. All the libraries were sequenced using HiSeq2500 Illumina in paired read mode creating reads with a length of 150 bp. GEO Accession:GSM3732419 RIP-Seq TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina HiSeq 2500   SRP162876     RIP_0hpf_rep2_R1.fastq.gz RIP_0hpf_rep2_R2.fastq.gz fastq fastq 14319153300.0 47730511.0 GSM3732419 r1 0:150 1:150 A:3633266473;C:3581763715;G:3602109377;T:3500497801;N:1515934 150 150     3633266473 3581763715 3602109377 3500497801 1515934 SRX5717512 SRS4655932 SRA786939 GEO Beijing Institute of Genomics (BIG) of Chinese Academy of Sciences (CAS) 2 0.96309 0.96299 0.09978 0.09922 0.80012 0.80635 0.7641 0.76535 150 150 B B biological fallback assumption illumina hiseq_era full_length random_priming smarter bulk clip iclip   China 2019-04-22 Zygote Embryo Whole Organism All anatomical structures
49546 49546 SRR8936998 SRX5717511 SRS4655931 SRP162876 PRJNA493828 Ybx1 binding sites and Ybx1 binding m5C sites in zebrafish embryo sample. GSE120646 Other The maternal to zygotic transition MZT is a conserved and fundamental process during which the embryo undergoes dramatic reprogramming to convert maternal environment to embryonic driven programing. However how the maternally supplied transcripts are dynamically regulated during MZT remains largely unknown. Herein through genome wide profiling of RNA 5 methylcytosine m5C in zebrafish early embryos we show that m5C methylated maternal mRNAs display higher stability during MZT. We identify that the Y box binding protein 1 Ybx1 prefers to recognizing m5C modified mRNAs through p p interaction with a key residue Trp45 in its cold shock domain CSD which plays essential roles in maternal mRNA stability and early embryogenesis of zebrafish. Cooperated with an mRNA stabilizer Pabpc1a Ybx1 promotes the stability of its target mRNAs in an m5C dependent manner. Our study demonstrates a novel mechanism of RNA m5C methylation regulated maternal mRNA stability during zebrafish MZT highlighting the critical role of m5C mRNA methylation in early development. Overall design: Examination of Ybx1 binding sites and Ybx1 binding m5C sites in zebrafish embryo. RIP seq: Briefly 500 zebrafish embryos at shield stage 4 hpf were resuspended with 2 ml lysis buffer 150 mM KCl 10 mM HEPES pH 7.6 2 mM EDTA 0.5% NP 40 0.5 mM DTT 1:100 protease inhibitor cocktail 0.4 U/μl RNasin by rotating at 4°C for 30 min and centrifuged at 15 000 g for 15 min. The supernatant was collected and pre cleared with protein A Dynabeads by rotation at 4°C for 1 h. 50 μl pre cleared embryo lysate was saved as Input and the left were incubated with affinity purified rabbit polyclonal anti Ybx1 antibody prepared by AbMax China and 30 μl Protein A Dynabeads for 4 h at 4°C or overnight. post washing with1 ml ice cold NT2 buffer 200 mM NaCl 50 mM HEPES pH 7.6 2 mM EDTA 0.05% NP 40 0.5 mM DTT 0.4U/µl RNasin for eight times and once with 1ml ice cold 1× PK buffer 100 mM Tris HCl pH 7.4 50 mM NaCl 10 mM EDTA 0.2% SDS the beads was treated i… parent bioproject:PRJNA534030     RIP 0hpf rep1 GSM3732418   source name:RIP 0hpf|strain:AB strain|age:0 hpf|tissue:whole embryo|rip antibody:rabbit polyclonal anti Ybx1 antibody RIP 0hpf rep1 Reads were aligned to the zv9 genome assembly using TopHat v2.1.1. MACS2 v2.1.1 were used for the peak calling and peaks were annotated by applying BEDTools’ intersectBed. Genome build: zv9 Supplementary files format and content: Ybx1 binding sites in two biological replicates. RIP 0hpf   Total RNA was isolated from zebrafish embryos harvested at different stages using TRIzol® Reagent Ambion. mRNA was extracted with using Dynabeads® mRNA Purification Kit Ambion and subjected to TURBO™ DNase Invitrogen treatment at 37°C for 30 min and ethanol precipitation. Thus purified mRNA was used for RNA BisSeq library construction. RNA was extracted and subjected to library construction using Smarter smRNA Seq kit Takara. All the libraries were sequenced using HiSeq2500 Illumina in paired read mode creating reads with a length of 150 bp.   strain:AB strain|age:0 hpf|tissue:whole embryo|rip antibody:rabbit polyclonal anti Ybx1 antibody GSM3732418 GSM3732418: RIP 0hpf rep1; Danio rerio; RIP Seq GSM3732418   1 Total RNA was isolated from zebrafish embryos harvested at different stages using TRIzol® Reagent Ambion. mRNA was extracted with using Dynabeads® mRNA Purification Kit Ambion and subjected to TURBO™ DNase Invitrogen treatment at 37°C for 30 min and ethanol precipitation. Thus purified mRNA was used for RNA BisSeq library construction. RNA was extracted and subjected to library construction using Smarter smRNA Seq kit Takara. All the libraries were sequenced using HiSeq2500 Illumina in paired read mode creating reads with a length of 150 bp. GEO Accession:GSM3732418 RIP-Seq TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina HiSeq 2500   SRP162876     RIP_0hpf_rep1_R1.fastq.gz RIP_0hpf_rep1_R2.fastq.gz fastq fastq 7807878000.0 26026260.0 GSM3732418 r1 0:150 1:150 A:1698013251;C:2200605244;G:2305251364;T:1602824248;N:1183893 150 150     1698013251 2200605244 2305251364 1602824248 1183893 SRX5717511 SRS4655931 SRA786939 GEO Beijing Institute of Genomics (BIG) of Chinese Academy of Sciences (CAS) 2 0.89154 0.89105 0.26373 0.26886 0.82477 0.827 0.79125 0.77711 150 150 B B biological fallback assumption illumina hiseq_era full_length random_priming smarter bulk clip iclip   China 2019-04-22 Zygote Embryo Whole Organism All anatomical structures
66194 66194 SRR16096233 SRX12382357 SRS10353019 SRP339082 PRJNA766854 RNA m6A methylome from zebrafish zygotes mouse liver polyA selected RNAs mouse embryonic stem cells spike in RNA controls and single mouse oocytes and embryos GSE184893 Other N6 methyladenosine m6A is a prevalent RNA modification that has key roles in distinct biological processes including meiosis and early embryo development. Despite advances in methodology for m6A mapping the applicability of current methods is limited by the need for large amounts of input material. We develop a sensitive picogram scale m6A RNA immunoprecipitation and sequencing picoMeRIP seq method also suitable for single cell scMeRIP seq. We validate the down scaling of the method by profiling m6A from picogram amounts of mouse liver polyA selected RNA mouse embryonic stem cells and zebrafish zygotes. We provide proof of principle of m6A profiling in single mouse oocytes and preimplantation embryos. Our work opens a new avenue for studying m6A in single cells and scarce cell types in a transcriptome wide manner. Overall design: Transcriptome wide m6A maps were generated from zebrafish and mouse samples as below. 11 zebrafish zygote samples: 3 biological replicates for 10 zygotes 3 biological replicates for 5 zygotes and 5 biological replicates for single zygote. 6 mouse liver polyA selected RNA samples: 10 ng 1 ng and 100 pg; 2 biological replicates for each amount. 9 mouse embryonic stem cell R1 cell line from ATCC SCRC 1011 samples: 1000 cells 100 cells and 10 cells; 3 biological replicates for each amount. 3 spike in RNA control add 0.001 fmol Gaussia luciferase/GLuc with m6A modification and 0.001 fmol Cypridina luciferase/CLuc without xxx modification into the 12 ng polyA selected RNAs related samples: 12 ng polyA selected RNA from mouse liver; 6 ng polyA selected RNA from mouse liver and 6 ng polyA selected RNA from wild type/WT mouse embryonic stem cells from Geula S. et al. Science 2015; and 6 ng polyA selected RNA from mouse liver and 6 ng polyA selected RNA from Mettl3 knock out/KO mouse embryonic stem cells from Geula S. et al. Science 2015. 12 single mouse oocyte and embryo samples: oocytes at germinal vesicle GV and metaphase II MII stages and embryos at zygote 2 cell 8 cell and…   pubmed:37349523   ZF zygote IP 1cell Rep5 GSM5599725   tissue:ZF zygote IP 1cell|developmental stage:Zygote|molecule subtype:rRNA free RNA ZF zygote IP 1cell Rep5 Quality of raw reads was assessed using FastQC v0.11.8 and sequencing adapters were trimmed using Cutadapt v1.8.1 if clear adapter signal was detected at the three primeend of the reads. There is no adapter removal for zebrafish zygote samples GSM5599713 GSM5599714 GSM5599715 GSM5599716 GSM5599717 GSM5599718 GSM5599719 GSM5599720 GSM5599721 GSM5599722 GSM5599723 GSM5599724 GSM5599725 mouse polyA selected RNA samples GSM5599726 GSM5599727 GSM5599728 GSM5599729 GSM5599730 GSM5599731 GSM5599732 GSM5599733 and mouse MII oocyte Input sample GSM5599734. Trimmed reads were aligned to the reference genome mm10 for mouse danRer11 for zebrafish using HISAT2 v2.1.0 with the parameter “ 5 8 no mixed no discordant”. Multiply aligned reads were discarded as indicated by HISAT2. PCR duplicates were removed using Samtools v1.9. The read pairs that overlapped with ribosomal RNAs were removed using BEDTools v2.28.0. Genome coverage bigWig files bin size = 10 bp normalized by RPKM were generated by deepTools v3.2.0 bamCoverage with the parameter “ bs 10 normalizeUsing RPKM”. Genome build: mm10 for mouse danRer11 for zebrafish Supplementary files format and content: The bigWig files are generated by deepTools v3.2.0 bamCoverage with the bin size=10bp and normalized by RPKM. ZF zygote IP 1cell For mouse GV oocyte collection 8 wpf C57BL6/N females were injected with 5 units U of pregnant mare serum gonadotropin PMSG. post 48 hours ovaries were dissected and oocytes were isolated by puncturing the follicles. The procedure was carried out in M2 medium Sigma supplemented with 0.2 mM of the cyclic nucleotide phosphodiesterase inhibitor 3 Isobutyl 1 methylxanthine IBMX Sigma to prevent the oocytes from further progress to germinal vesicle breakdown GVBD. The cumulus cells were gently removed by pipetting and the oocytes were briefly exposed to acidic Tyrode's solution Sigma to remove the zona pellucida followed by three washes in M2 medium. For mouse MII oocyte collection 4 wpf 5 wpf C57BL6/N females were injected with 5 U of PMSG followed by 5 U of human chorionic gonadotropin hCG 45 hours later. The oviducts were dissected 20 22 hours later and transferred to a clean dish containing M2 medium. The oviduct ampulla was identified under a stereomicroscope to isolate MII oocytes containing the cumulus mass. The oocytes were treated with 0.3 mg/ml hyaluronidase dissolved in M2 medium to remove the cumulus cells and then exposed to acidic Tyrode's solution for a few seconds to remove the zona pellucida followed by three washes in M2 medium. For mouse early embryo collection 8 wpf C57BL/6N females were superovulated by hormone injection 5 U PMSG followed by 5 U hCG 45 hours later and transferred to cages with 8 wpf C57BL/6N males for mating. At 27 28 hours zygote 39 43 hours 2 cell 68 70 hours 8 cell and 92 94 hours blastocyst post hCG administration the female mice were euthanized by cervical dislocation. Embryos were flushed from the reproductive tract into HEPES buffered CZB medium then transferred to acidic Tyrode's solution for a few seconds to remove the zona pellucida followed by three washes in M2 medium. For zebrafish bulk zygote samples total RNA was extracted from 100 zebrafish zygotes using TRIzol™ Reagent and eluated in 100 μl of RNase free water. Then 10 μl and 5 μl samples were taken for 10 zygote and 5 zygote samples respectively and volumes were adjusted to 12 μl with nuclease free water. For zebrafish single zygote samples single zygote was manually picked and distributed into 12 μl of 1X Lysis buffer Takara. For the polyA selected RNA samples of mouse liver and embryonic stem cell WT and Mettl3 KO total RNA was extracted using TRIzol™ Reagent Thermo Fisher Scientific and PolyA+ RNA was selected twice by Dynabeads™ mRNA Purification Kit Thermo Fisher Scientific. For mouse embryonic stem cell samples the cells were sorted into 12 μl 1X Lysis buffer using BD FACSMelody™ Cell Sorter BD Biosciences. For mouse oocyte and embryo samples post three washes in M2 medium oocytes and embryos were manually picked and put into 12 μl 1X Lysis buffer. For picoMeRIP seq experiment NEBNext® rRNA Depletion Kit NEB was used to delete DNA and rRNA; RNA samples were sheared by sonication incubated with the anti m6A antibody from Millipore ABE572 and precipitated using ethanol. SMART Seq® Stranded Kit Takara Cat. # 634442 was used to prepare RNA sequencing library. Libraries were quantified with KAPA Library Quantification Kits Roche and the size distribution was checked using TapeStation D1000 ScreenTape Agilent Technologies.   developmental stage:Zygote|molecule subtype:rRNA free RNA GSM5599725 GSM5599725: ZF zygote IP 1cell Rep5; Danio rerio; RIP Seq GSM5599725   1 For mouse liver total RNA was extracted using TRIzol™ Reagent Thermo Fisher Scientific and PolyA+ RNA was selected twice by Dynabeads™ mRNA Purification Kit Thermo Fisher Scientific. For zebrafish zygote mouse MII oocyte and mouse blastocyst samples the single cells were manually picked and sorted into 12 μl 1X lysis buffer Takara; NEBNext® rRNA Depletion Kit NEB was used to delete DNA and rRNA; RNA samples were sonicated for 2 × 30 seconds incubated with the anti m6A antibody from Millipore ABE572 and precipitated using ethanol. SMART Seq® Stranded Kit Takara Cat. # 634442 was used to prepare RNA sequencing library. Libraries were quantified with KAPA Library Quantification Kits Roche and the size distribution was checked using TapeStation D1000 ScreenTape Agilent Technologies. GEO Accession:GSM5599725 RIP-Seq TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina NovaSeq 6000   SRP339082   loader:fastq load.py ZF_zygote_IP_1cell_Rep5_R1.fastq.gz ZF_zygote_IP_1cell_Rep5_R2.fastq.gz fastq fastq 3660517860.0 35887430.0 GSM5599725 r1 0:51 1:51 A:858138600;C:924733544;G:1002150083;T:875349961;N:145672 51 51     858138600 924733544 1002150083 875349961 145672 SRX12382357 SRS10353019 SRA1301707 GEO Room 2035, Palmer Commons, Department of Computational Medicine and Bioinformatics, University of Michigan 2 0.83384 0.82667 0.09687 0.11892 0.79076 0.79326 0.63739 0.60941 51 51 B B biological fallback assumption illumina novaseq_era 3prime poly_a nebnext bulk bulk bulk   United States 2021-09-28 Zygote Embryo Oocyte Reproductive System
66195 66195 SRR16096232 SRX12382356 SRS10353018 SRP339082 PRJNA766854 RNA m6A methylome from zebrafish zygotes mouse liver polyA selected RNAs mouse embryonic stem cells spike in RNA controls and single mouse oocytes and embryos GSE184893 Other N6 methyladenosine m6A is a prevalent RNA modification that has key roles in distinct biological processes including meiosis and early embryo development. Despite advances in methodology for m6A mapping the applicability of current methods is limited by the need for large amounts of input material. We develop a sensitive picogram scale m6A RNA immunoprecipitation and sequencing picoMeRIP seq method also suitable for single cell scMeRIP seq. We validate the down scaling of the method by profiling m6A from picogram amounts of mouse liver polyA selected RNA mouse embryonic stem cells and zebrafish zygotes. We provide proof of principle of m6A profiling in single mouse oocytes and preimplantation embryos. Our work opens a new avenue for studying m6A in single cells and scarce cell types in a transcriptome wide manner. Overall design: Transcriptome wide m6A maps were generated from zebrafish and mouse samples as below. 11 zebrafish zygote samples: 3 biological replicates for 10 zygotes 3 biological replicates for 5 zygotes and 5 biological replicates for single zygote. 6 mouse liver polyA selected RNA samples: 10 ng 1 ng and 100 pg; 2 biological replicates for each amount. 9 mouse embryonic stem cell R1 cell line from ATCC SCRC 1011 samples: 1000 cells 100 cells and 10 cells; 3 biological replicates for each amount. 3 spike in RNA control add 0.001 fmol Gaussia luciferase/GLuc with m6A modification and 0.001 fmol Cypridina luciferase/CLuc without xxx modification into the 12 ng polyA selected RNAs related samples: 12 ng polyA selected RNA from mouse liver; 6 ng polyA selected RNA from mouse liver and 6 ng polyA selected RNA from wild type/WT mouse embryonic stem cells from Geula S. et al. Science 2015; and 6 ng polyA selected RNA from mouse liver and 6 ng polyA selected RNA from Mettl3 knock out/KO mouse embryonic stem cells from Geula S. et al. Science 2015. 12 single mouse oocyte and embryo samples: oocytes at germinal vesicle GV and metaphase II MII stages and embryos at zygote 2 cell 8 cell and…   pubmed:37349523   ZF zygote IP 1cell Rep4 GSM5599724   tissue:ZF zygote IP 1cell|developmental stage:Zygote|molecule subtype:rRNA free RNA ZF zygote IP 1cell Rep4 Quality of raw reads was assessed using FastQC v0.11.8 and sequencing adapters were trimmed using Cutadapt v1.8.1 if clear adapter signal was detected at the three primeend of the reads. There is no adapter removal for zebrafish zygote samples GSM5599713 GSM5599714 GSM5599715 GSM5599716 GSM5599717 GSM5599718 GSM5599719 GSM5599720 GSM5599721 GSM5599722 GSM5599723 GSM5599724 GSM5599725 mouse polyA selected RNA samples GSM5599726 GSM5599727 GSM5599728 GSM5599729 GSM5599730 GSM5599731 GSM5599732 GSM5599733 and mouse MII oocyte Input sample GSM5599734. Trimmed reads were aligned to the reference genome mm10 for mouse danRer11 for zebrafish using HISAT2 v2.1.0 with the parameter “ 5 8 no mixed no discordant”. Multiply aligned reads were discarded as indicated by HISAT2. PCR duplicates were removed using Samtools v1.9. The read pairs that overlapped with ribosomal RNAs were removed using BEDTools v2.28.0. Genome coverage bigWig files bin size = 10 bp normalized by RPKM were generated by deepTools v3.2.0 bamCoverage with the parameter “ bs 10 normalizeUsing RPKM”. Genome build: mm10 for mouse danRer11 for zebrafish Supplementary files format and content: The bigWig files are generated by deepTools v3.2.0 bamCoverage with the bin size=10bp and normalized by RPKM. ZF zygote IP 1cell For mouse GV oocyte collection 8 wpf C57BL6/N females were injected with 5 units U of pregnant mare serum gonadotropin PMSG. post 48 hours ovaries were dissected and oocytes were isolated by puncturing the follicles. The procedure was carried out in M2 medium Sigma supplemented with 0.2 mM of the cyclic nucleotide phosphodiesterase inhibitor 3 Isobutyl 1 methylxanthine IBMX Sigma to prevent the oocytes from further progress to germinal vesicle breakdown GVBD. The cumulus cells were gently removed by pipetting and the oocytes were briefly exposed to acidic Tyrode's solution Sigma to remove the zona pellucida followed by three washes in M2 medium. For mouse MII oocyte collection 4 wpf 5 wpf C57BL6/N females were injected with 5 U of PMSG followed by 5 U of human chorionic gonadotropin hCG 45 hours later. The oviducts were dissected 20 22 hours later and transferred to a clean dish containing M2 medium. The oviduct ampulla was identified under a stereomicroscope to isolate MII oocytes containing the cumulus mass. The oocytes were treated with 0.3 mg/ml hyaluronidase dissolved in M2 medium to remove the cumulus cells and then exposed to acidic Tyrode's solution for a few seconds to remove the zona pellucida followed by three washes in M2 medium. For mouse early embryo collection 8 wpf C57BL/6N females were superovulated by hormone injection 5 U PMSG followed by 5 U hCG 45 hours later and transferred to cages with 8 wpf C57BL/6N males for mating. At 27 28 hours zygote 39 43 hours 2 cell 68 70 hours 8 cell and 92 94 hours blastocyst post hCG administration the female mice were euthanized by cervical dislocation. Embryos were flushed from the reproductive tract into HEPES buffered CZB medium then transferred to acidic Tyrode's solution for a few seconds to remove the zona pellucida followed by three washes in M2 medium. For zebrafish bulk zygote samples total RNA was extracted from 100 zebrafish zygotes using TRIzol™ Reagent and eluated in 100 μl of RNase free water. Then 10 μl and 5 μl samples were taken for 10 zygote and 5 zygote samples respectively and volumes were adjusted to 12 μl with nuclease free water. For zebrafish single zygote samples single zygote was manually picked and distributed into 12 μl of 1X Lysis buffer Takara. For the polyA selected RNA samples of mouse liver and embryonic stem cell WT and Mettl3 KO total RNA was extracted using TRIzol™ Reagent Thermo Fisher Scientific and PolyA+ RNA was selected twice by Dynabeads™ mRNA Purification Kit Thermo Fisher Scientific. For mouse embryonic stem cell samples the cells were sorted into 12 μl 1X Lysis buffer using BD FACSMelody™ Cell Sorter BD Biosciences. For mouse oocyte and embryo samples post three washes in M2 medium oocytes and embryos were manually picked and put into 12 μl 1X Lysis buffer. For picoMeRIP seq experiment NEBNext® rRNA Depletion Kit NEB was used to delete DNA and rRNA; RNA samples were sheared by sonication incubated with the anti m6A antibody from Millipore ABE572 and precipitated using ethanol. SMART Seq® Stranded Kit Takara Cat. # 634442 was used to prepare RNA sequencing library. Libraries were quantified with KAPA Library Quantification Kits Roche and the size distribution was checked using TapeStation D1000 ScreenTape Agilent Technologies.   developmental stage:Zygote|molecule subtype:rRNA free RNA GSM5599724 GSM5599724: ZF zygote IP 1cell Rep4; Danio rerio; RIP Seq GSM5599724   1 For mouse liver total RNA was extracted using TRIzol™ Reagent Thermo Fisher Scientific and PolyA+ RNA was selected twice by Dynabeads™ mRNA Purification Kit Thermo Fisher Scientific. For zebrafish zygote mouse MII oocyte and mouse blastocyst samples the single cells were manually picked and sorted into 12 μl 1X lysis buffer Takara; NEBNext® rRNA Depletion Kit NEB was used to delete DNA and rRNA; RNA samples were sonicated for 2 × 30 seconds incubated with the anti m6A antibody from Millipore ABE572 and precipitated using ethanol. SMART Seq® Stranded Kit Takara Cat. # 634442 was used to prepare RNA sequencing library. Libraries were quantified with KAPA Library Quantification Kits Roche and the size distribution was checked using TapeStation D1000 ScreenTape Agilent Technologies. GEO Accession:GSM5599724 RIP-Seq TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina NovaSeq 6000   SRP339082   loader:fastq load.py ZF_zygote_IP_1cell_Rep4_R1.fastq.gz ZF_zygote_IP_1cell_Rep4_R2.fastq.gz fastq fastq 3660205842.0 35884371.0 GSM5599724 r1 0:51 1:51 A:895738659;C:880838836;G:971118499;T:912364119;N:145729 51 51     895738659 880838836 971118499 912364119 145729 SRX12382356 SRS10353018 SRA1301707 GEO Room 2035, Palmer Commons, Department of Computational Medicine and Bioinformatics, University of Michigan 2 0.87258 0.86932 0.06573 0.07958 0.78044 0.78374 0.55531 0.5462 51 51 B B biological fallback assumption illumina novaseq_era 3prime poly_a nebnext bulk bulk bulk   United States 2021-09-28 Zygote Embryo Oocyte Reproductive System
66196 66196 SRR16096231 SRX12382355 SRS10353016 SRP339082 PRJNA766854 RNA m6A methylome from zebrafish zygotes mouse liver polyA selected RNAs mouse embryonic stem cells spike in RNA controls and single mouse oocytes and embryos GSE184893 Other N6 methyladenosine m6A is a prevalent RNA modification that has key roles in distinct biological processes including meiosis and early embryo development. Despite advances in methodology for m6A mapping the applicability of current methods is limited by the need for large amounts of input material. We develop a sensitive picogram scale m6A RNA immunoprecipitation and sequencing picoMeRIP seq method also suitable for single cell scMeRIP seq. We validate the down scaling of the method by profiling m6A from picogram amounts of mouse liver polyA selected RNA mouse embryonic stem cells and zebrafish zygotes. We provide proof of principle of m6A profiling in single mouse oocytes and preimplantation embryos. Our work opens a new avenue for studying m6A in single cells and scarce cell types in a transcriptome wide manner. Overall design: Transcriptome wide m6A maps were generated from zebrafish and mouse samples as below. 11 zebrafish zygote samples: 3 biological replicates for 10 zygotes 3 biological replicates for 5 zygotes and 5 biological replicates for single zygote. 6 mouse liver polyA selected RNA samples: 10 ng 1 ng and 100 pg; 2 biological replicates for each amount. 9 mouse embryonic stem cell R1 cell line from ATCC SCRC 1011 samples: 1000 cells 100 cells and 10 cells; 3 biological replicates for each amount. 3 spike in RNA control add 0.001 fmol Gaussia luciferase/GLuc with m6A modification and 0.001 fmol Cypridina luciferase/CLuc without xxx modification into the 12 ng polyA selected RNAs related samples: 12 ng polyA selected RNA from mouse liver; 6 ng polyA selected RNA from mouse liver and 6 ng polyA selected RNA from wild type/WT mouse embryonic stem cells from Geula S. et al. Science 2015; and 6 ng polyA selected RNA from mouse liver and 6 ng polyA selected RNA from Mettl3 knock out/KO mouse embryonic stem cells from Geula S. et al. Science 2015. 12 single mouse oocyte and embryo samples: oocytes at germinal vesicle GV and metaphase II MII stages and embryos at zygote 2 cell 8 cell and…   pubmed:37349523   ZF zygote IP 1cell Rep3 GSM5599723   tissue:ZF zygote IP 1cell|developmental stage:Zygote|molecule subtype:rRNA free RNA ZF zygote IP 1cell Rep3 Quality of raw reads was assessed using FastQC v0.11.8 and sequencing adapters were trimmed using Cutadapt v1.8.1 if clear adapter signal was detected at the three primeend of the reads. There is no adapter removal for zebrafish zygote samples GSM5599713 GSM5599714 GSM5599715 GSM5599716 GSM5599717 GSM5599718 GSM5599719 GSM5599720 GSM5599721 GSM5599722 GSM5599723 GSM5599724 GSM5599725 mouse polyA selected RNA samples GSM5599726 GSM5599727 GSM5599728 GSM5599729 GSM5599730 GSM5599731 GSM5599732 GSM5599733 and mouse MII oocyte Input sample GSM5599734. Trimmed reads were aligned to the reference genome mm10 for mouse danRer11 for zebrafish using HISAT2 v2.1.0 with the parameter “ 5 8 no mixed no discordant”. Multiply aligned reads were discarded as indicated by HISAT2. PCR duplicates were removed using Samtools v1.9. The read pairs that overlapped with ribosomal RNAs were removed using BEDTools v2.28.0. Genome coverage bigWig files bin size = 10 bp normalized by RPKM were generated by deepTools v3.2.0 bamCoverage with the parameter “ bs 10 normalizeUsing RPKM”. Genome build: mm10 for mouse danRer11 for zebrafish Supplementary files format and content: The bigWig files are generated by deepTools v3.2.0 bamCoverage with the bin size=10bp and normalized by RPKM. ZF zygote IP 1cell For mouse GV oocyte collection 8 wpf C57BL6/N females were injected with 5 units U of pregnant mare serum gonadotropin PMSG. post 48 hours ovaries were dissected and oocytes were isolated by puncturing the follicles. The procedure was carried out in M2 medium Sigma supplemented with 0.2 mM of the cyclic nucleotide phosphodiesterase inhibitor 3 Isobutyl 1 methylxanthine IBMX Sigma to prevent the oocytes from further progress to germinal vesicle breakdown GVBD. The cumulus cells were gently removed by pipetting and the oocytes were briefly exposed to acidic Tyrode's solution Sigma to remove the zona pellucida followed by three washes in M2 medium. For mouse MII oocyte collection 4 wpf 5 wpf C57BL6/N females were injected with 5 U of PMSG followed by 5 U of human chorionic gonadotropin hCG 45 hours later. The oviducts were dissected 20 22 hours later and transferred to a clean dish containing M2 medium. The oviduct ampulla was identified under a stereomicroscope to isolate MII oocytes containing the cumulus mass. The oocytes were treated with 0.3 mg/ml hyaluronidase dissolved in M2 medium to remove the cumulus cells and then exposed to acidic Tyrode's solution for a few seconds to remove the zona pellucida followed by three washes in M2 medium. For mouse early embryo collection 8 wpf C57BL/6N females were superovulated by hormone injection 5 U PMSG followed by 5 U hCG 45 hours later and transferred to cages with 8 wpf C57BL/6N males for mating. At 27 28 hours zygote 39 43 hours 2 cell 68 70 hours 8 cell and 92 94 hours blastocyst post hCG administration the female mice were euthanized by cervical dislocation. Embryos were flushed from the reproductive tract into HEPES buffered CZB medium then transferred to acidic Tyrode's solution for a few seconds to remove the zona pellucida followed by three washes in M2 medium. For zebrafish bulk zygote samples total RNA was extracted from 100 zebrafish zygotes using TRIzol™ Reagent and eluated in 100 μl of RNase free water. Then 10 μl and 5 μl samples were taken for 10 zygote and 5 zygote samples respectively and volumes were adjusted to 12 μl with nuclease free water. For zebrafish single zygote samples single zygote was manually picked and distributed into 12 μl of 1X Lysis buffer Takara. For the polyA selected RNA samples of mouse liver and embryonic stem cell WT and Mettl3 KO total RNA was extracted using TRIzol™ Reagent Thermo Fisher Scientific and PolyA+ RNA was selected twice by Dynabeads™ mRNA Purification Kit Thermo Fisher Scientific. For mouse embryonic stem cell samples the cells were sorted into 12 μl 1X Lysis buffer using BD FACSMelody™ Cell Sorter BD Biosciences. For mouse oocyte and embryo samples post three washes in M2 medium oocytes and embryos were manually picked and put into 12 μl 1X Lysis buffer. For picoMeRIP seq experiment NEBNext® rRNA Depletion Kit NEB was used to delete DNA and rRNA; RNA samples were sheared by sonication incubated with the anti m6A antibody from Millipore ABE572 and precipitated using ethanol. SMART Seq® Stranded Kit Takara Cat. # 634442 was used to prepare RNA sequencing library. Libraries were quantified with KAPA Library Quantification Kits Roche and the size distribution was checked using TapeStation D1000 ScreenTape Agilent Technologies.   developmental stage:Zygote|molecule subtype:rRNA free RNA GSM5599723 GSM5599723: ZF zygote IP 1cell Rep3; Danio rerio; RIP Seq GSM5599723   1 For mouse liver total RNA was extracted using TRIzol™ Reagent Thermo Fisher Scientific and PolyA+ RNA was selected twice by Dynabeads™ mRNA Purification Kit Thermo Fisher Scientific. For zebrafish zygote mouse MII oocyte and mouse blastocyst samples the single cells were manually picked and sorted into 12 μl 1X lysis buffer Takara; NEBNext® rRNA Depletion Kit NEB was used to delete DNA and rRNA; RNA samples were sonicated for 2 × 30 seconds incubated with the anti m6A antibody from Millipore ABE572 and precipitated using ethanol. SMART Seq® Stranded Kit Takara Cat. # 634442 was used to prepare RNA sequencing library. Libraries were quantified with KAPA Library Quantification Kits Roche and the size distribution was checked using TapeStation D1000 ScreenTape Agilent Technologies. GEO Accession:GSM5599723 RIP-Seq TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina NovaSeq 6000   SRP339082   loader:fastq load.py ZF_zygote_IP_1cell_Rep3_R1.fastq.gz ZF_zygote_IP_1cell_Rep3_R2.fastq.gz fastq fastq 2732409048.0 26788324.0 GSM5599723 r1 0:51 1:51 A:669345195;C:656715671;G:724415204;T:681825149;N:107829 51 51     669345195 656715671 724415204 681825149 107829 SRX12382355 SRS10353016 SRA1301707 GEO Room 2035, Palmer Commons, Department of Computational Medicine and Bioinformatics, University of Michigan 2 0.85204 0.83607 0.06526 0.08046 0.78535 0.78748 0.60467 0.50165 51 51 B B biological fallback assumption illumina novaseq_era 3prime poly_a nebnext bulk bulk bulk   United States 2021-09-28 Zygote Embryo Oocyte Reproductive System
66197 66197 SRR16096230 SRX12382354 SRS10353017 SRP339082 PRJNA766854 RNA m6A methylome from zebrafish zygotes mouse liver polyA selected RNAs mouse embryonic stem cells spike in RNA controls and single mouse oocytes and embryos GSE184893 Other N6 methyladenosine m6A is a prevalent RNA modification that has key roles in distinct biological processes including meiosis and early embryo development. Despite advances in methodology for m6A mapping the applicability of current methods is limited by the need for large amounts of input material. We develop a sensitive picogram scale m6A RNA immunoprecipitation and sequencing picoMeRIP seq method also suitable for single cell scMeRIP seq. We validate the down scaling of the method by profiling m6A from picogram amounts of mouse liver polyA selected RNA mouse embryonic stem cells and zebrafish zygotes. We provide proof of principle of m6A profiling in single mouse oocytes and preimplantation embryos. Our work opens a new avenue for studying m6A in single cells and scarce cell types in a transcriptome wide manner. Overall design: Transcriptome wide m6A maps were generated from zebrafish and mouse samples as below. 11 zebrafish zygote samples: 3 biological replicates for 10 zygotes 3 biological replicates for 5 zygotes and 5 biological replicates for single zygote. 6 mouse liver polyA selected RNA samples: 10 ng 1 ng and 100 pg; 2 biological replicates for each amount. 9 mouse embryonic stem cell R1 cell line from ATCC SCRC 1011 samples: 1000 cells 100 cells and 10 cells; 3 biological replicates for each amount. 3 spike in RNA control add 0.001 fmol Gaussia luciferase/GLuc with m6A modification and 0.001 fmol Cypridina luciferase/CLuc without xxx modification into the 12 ng polyA selected RNAs related samples: 12 ng polyA selected RNA from mouse liver; 6 ng polyA selected RNA from mouse liver and 6 ng polyA selected RNA from wild type/WT mouse embryonic stem cells from Geula S. et al. Science 2015; and 6 ng polyA selected RNA from mouse liver and 6 ng polyA selected RNA from Mettl3 knock out/KO mouse embryonic stem cells from Geula S. et al. Science 2015. 12 single mouse oocyte and embryo samples: oocytes at germinal vesicle GV and metaphase II MII stages and embryos at zygote 2 cell 8 cell and…   pubmed:37349523   ZF zygote IP 1cell Rep2 GSM5599722   tissue:ZF zygote IP 1cell|developmental stage:Zygote|molecule subtype:rRNA free RNA ZF zygote IP 1cell Rep2 Quality of raw reads was assessed using FastQC v0.11.8 and sequencing adapters were trimmed using Cutadapt v1.8.1 if clear adapter signal was detected at the three primeend of the reads. There is no adapter removal for zebrafish zygote samples GSM5599713 GSM5599714 GSM5599715 GSM5599716 GSM5599717 GSM5599718 GSM5599719 GSM5599720 GSM5599721 GSM5599722 GSM5599723 GSM5599724 GSM5599725 mouse polyA selected RNA samples GSM5599726 GSM5599727 GSM5599728 GSM5599729 GSM5599730 GSM5599731 GSM5599732 GSM5599733 and mouse MII oocyte Input sample GSM5599734. Trimmed reads were aligned to the reference genome mm10 for mouse danRer11 for zebrafish using HISAT2 v2.1.0 with the parameter “ 5 8 no mixed no discordant”. Multiply aligned reads were discarded as indicated by HISAT2. PCR duplicates were removed using Samtools v1.9. The read pairs that overlapped with ribosomal RNAs were removed using BEDTools v2.28.0. Genome coverage bigWig files bin size = 10 bp normalized by RPKM were generated by deepTools v3.2.0 bamCoverage with the parameter “ bs 10 normalizeUsing RPKM”. Genome build: mm10 for mouse danRer11 for zebrafish Supplementary files format and content: The bigWig files are generated by deepTools v3.2.0 bamCoverage with the bin size=10bp and normalized by RPKM. ZF zygote IP 1cell For mouse GV oocyte collection 8 wpf C57BL6/N females were injected with 5 units U of pregnant mare serum gonadotropin PMSG. post 48 hours ovaries were dissected and oocytes were isolated by puncturing the follicles. The procedure was carried out in M2 medium Sigma supplemented with 0.2 mM of the cyclic nucleotide phosphodiesterase inhibitor 3 Isobutyl 1 methylxanthine IBMX Sigma to prevent the oocytes from further progress to germinal vesicle breakdown GVBD. The cumulus cells were gently removed by pipetting and the oocytes were briefly exposed to acidic Tyrode's solution Sigma to remove the zona pellucida followed by three washes in M2 medium. For mouse MII oocyte collection 4 wpf 5 wpf C57BL6/N females were injected with 5 U of PMSG followed by 5 U of human chorionic gonadotropin hCG 45 hours later. The oviducts were dissected 20 22 hours later and transferred to a clean dish containing M2 medium. The oviduct ampulla was identified under a stereomicroscope to isolate MII oocytes containing the cumulus mass. The oocytes were treated with 0.3 mg/ml hyaluronidase dissolved in M2 medium to remove the cumulus cells and then exposed to acidic Tyrode's solution for a few seconds to remove the zona pellucida followed by three washes in M2 medium. For mouse early embryo collection 8 wpf C57BL/6N females were superovulated by hormone injection 5 U PMSG followed by 5 U hCG 45 hours later and transferred to cages with 8 wpf C57BL/6N males for mating. At 27 28 hours zygote 39 43 hours 2 cell 68 70 hours 8 cell and 92 94 hours blastocyst post hCG administration the female mice were euthanized by cervical dislocation. Embryos were flushed from the reproductive tract into HEPES buffered CZB medium then transferred to acidic Tyrode's solution for a few seconds to remove the zona pellucida followed by three washes in M2 medium. For zebrafish bulk zygote samples total RNA was extracted from 100 zebrafish zygotes using TRIzol™ Reagent and eluated in 100 μl of RNase free water. Then 10 μl and 5 μl samples were taken for 10 zygote and 5 zygote samples respectively and volumes were adjusted to 12 μl with nuclease free water. For zebrafish single zygote samples single zygote was manually picked and distributed into 12 μl of 1X Lysis buffer Takara. For the polyA selected RNA samples of mouse liver and embryonic stem cell WT and Mettl3 KO total RNA was extracted using TRIzol™ Reagent Thermo Fisher Scientific and PolyA+ RNA was selected twice by Dynabeads™ mRNA Purification Kit Thermo Fisher Scientific. For mouse embryonic stem cell samples the cells were sorted into 12 μl 1X Lysis buffer using BD FACSMelody™ Cell Sorter BD Biosciences. For mouse oocyte and embryo samples post three washes in M2 medium oocytes and embryos were manually picked and put into 12 μl 1X Lysis buffer. For picoMeRIP seq experiment NEBNext® rRNA Depletion Kit NEB was used to delete DNA and rRNA; RNA samples were sheared by sonication incubated with the anti m6A antibody from Millipore ABE572 and precipitated using ethanol. SMART Seq® Stranded Kit Takara Cat. # 634442 was used to prepare RNA sequencing library. Libraries were quantified with KAPA Library Quantification Kits Roche and the size distribution was checked using TapeStation D1000 ScreenTape Agilent Technologies.   developmental stage:Zygote|molecule subtype:rRNA free RNA GSM5599722 GSM5599722: ZF zygote IP 1cell Rep2; Danio rerio; RIP Seq GSM5599722   1 For mouse liver total RNA was extracted using TRIzol™ Reagent Thermo Fisher Scientific and PolyA+ RNA was selected twice by Dynabeads™ mRNA Purification Kit Thermo Fisher Scientific. For zebrafish zygote mouse MII oocyte and mouse blastocyst samples the single cells were manually picked and sorted into 12 μl 1X lysis buffer Takara; NEBNext® rRNA Depletion Kit NEB was used to delete DNA and rRNA; RNA samples were sonicated for 2 × 30 seconds incubated with the anti m6A antibody from Millipore ABE572 and precipitated using ethanol. SMART Seq® Stranded Kit Takara Cat. # 634442 was used to prepare RNA sequencing library. Libraries were quantified with KAPA Library Quantification Kits Roche and the size distribution was checked using TapeStation D1000 ScreenTape Agilent Technologies. GEO Accession:GSM5599722 RIP-Seq TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina NovaSeq 6000   SRP339082   loader:fastq load.py ZF_zygote_IP_1cell_Rep2_R1.fastq.gz ZF_zygote_IP_1cell_Rep2_R2.fastq.gz fastq fastq 2235668028.0 21918314.0 GSM5599722 r1 0:51 1:51 A:552522700;C:535947981;G:588168855;T:558939713;N:88779 51 51     552522700 535947981 588168855 558939713 88779 SRX12382354 SRS10353017 SRA1301707 GEO Room 2035, Palmer Commons, Department of Computational Medicine and Bioinformatics, University of Michigan 2 0.85597 0.85298 0.06663 0.07927 0.79058 0.79237 0.57626 0.56525 51 51 B B biological fallback assumption illumina novaseq_era 3prime poly_a nebnext bulk bulk bulk   United States 2021-09-28 Zygote Embryo Oocyte Reproductive System
66198 66198 SRR16096229 SRX12382353 SRS10353015 SRP339082 PRJNA766854 RNA m6A methylome from zebrafish zygotes mouse liver polyA selected RNAs mouse embryonic stem cells spike in RNA controls and single mouse oocytes and embryos GSE184893 Other N6 methyladenosine m6A is a prevalent RNA modification that has key roles in distinct biological processes including meiosis and early embryo development. Despite advances in methodology for m6A mapping the applicability of current methods is limited by the need for large amounts of input material. We develop a sensitive picogram scale m6A RNA immunoprecipitation and sequencing picoMeRIP seq method also suitable for single cell scMeRIP seq. We validate the down scaling of the method by profiling m6A from picogram amounts of mouse liver polyA selected RNA mouse embryonic stem cells and zebrafish zygotes. We provide proof of principle of m6A profiling in single mouse oocytes and preimplantation embryos. Our work opens a new avenue for studying m6A in single cells and scarce cell types in a transcriptome wide manner. Overall design: Transcriptome wide m6A maps were generated from zebrafish and mouse samples as below. 11 zebrafish zygote samples: 3 biological replicates for 10 zygotes 3 biological replicates for 5 zygotes and 5 biological replicates for single zygote. 6 mouse liver polyA selected RNA samples: 10 ng 1 ng and 100 pg; 2 biological replicates for each amount. 9 mouse embryonic stem cell R1 cell line from ATCC SCRC 1011 samples: 1000 cells 100 cells and 10 cells; 3 biological replicates for each amount. 3 spike in RNA control add 0.001 fmol Gaussia luciferase/GLuc with m6A modification and 0.001 fmol Cypridina luciferase/CLuc without xxx modification into the 12 ng polyA selected RNAs related samples: 12 ng polyA selected RNA from mouse liver; 6 ng polyA selected RNA from mouse liver and 6 ng polyA selected RNA from wild type/WT mouse embryonic stem cells from Geula S. et al. Science 2015; and 6 ng polyA selected RNA from mouse liver and 6 ng polyA selected RNA from Mettl3 knock out/KO mouse embryonic stem cells from Geula S. et al. Science 2015. 12 single mouse oocyte and embryo samples: oocytes at germinal vesicle GV and metaphase II MII stages and embryos at zygote 2 cell 8 cell and…   pubmed:37349523   ZF zygote IP 1cell Rep1 GSM5599721   tissue:ZF zygote IP 1cell|developmental stage:Zygote|molecule subtype:rRNA free RNA ZF zygote IP 1cell Rep1 Quality of raw reads was assessed using FastQC v0.11.8 and sequencing adapters were trimmed using Cutadapt v1.8.1 if clear adapter signal was detected at the three primeend of the reads. There is no adapter removal for zebrafish zygote samples GSM5599713 GSM5599714 GSM5599715 GSM5599716 GSM5599717 GSM5599718 GSM5599719 GSM5599720 GSM5599721 GSM5599722 GSM5599723 GSM5599724 GSM5599725 mouse polyA selected RNA samples GSM5599726 GSM5599727 GSM5599728 GSM5599729 GSM5599730 GSM5599731 GSM5599732 GSM5599733 and mouse MII oocyte Input sample GSM5599734. Trimmed reads were aligned to the reference genome mm10 for mouse danRer11 for zebrafish using HISAT2 v2.1.0 with the parameter “ 5 8 no mixed no discordant”. Multiply aligned reads were discarded as indicated by HISAT2. PCR duplicates were removed using Samtools v1.9. The read pairs that overlapped with ribosomal RNAs were removed using BEDTools v2.28.0. Genome coverage bigWig files bin size = 10 bp normalized by RPKM were generated by deepTools v3.2.0 bamCoverage with the parameter “ bs 10 normalizeUsing RPKM”. Genome build: mm10 for mouse danRer11 for zebrafish Supplementary files format and content: The bigWig files are generated by deepTools v3.2.0 bamCoverage with the bin size=10bp and normalized by RPKM. ZF zygote IP 1cell For mouse GV oocyte collection 8 wpf C57BL6/N females were injected with 5 units U of pregnant mare serum gonadotropin PMSG. post 48 hours ovaries were dissected and oocytes were isolated by puncturing the follicles. The procedure was carried out in M2 medium Sigma supplemented with 0.2 mM of the cyclic nucleotide phosphodiesterase inhibitor 3 Isobutyl 1 methylxanthine IBMX Sigma to prevent the oocytes from further progress to germinal vesicle breakdown GVBD. The cumulus cells were gently removed by pipetting and the oocytes were briefly exposed to acidic Tyrode's solution Sigma to remove the zona pellucida followed by three washes in M2 medium. For mouse MII oocyte collection 4 wpf 5 wpf C57BL6/N females were injected with 5 U of PMSG followed by 5 U of human chorionic gonadotropin hCG 45 hours later. The oviducts were dissected 20 22 hours later and transferred to a clean dish containing M2 medium. The oviduct ampulla was identified under a stereomicroscope to isolate MII oocytes containing the cumulus mass. The oocytes were treated with 0.3 mg/ml hyaluronidase dissolved in M2 medium to remove the cumulus cells and then exposed to acidic Tyrode's solution for a few seconds to remove the zona pellucida followed by three washes in M2 medium. For mouse early embryo collection 8 wpf C57BL/6N females were superovulated by hormone injection 5 U PMSG followed by 5 U hCG 45 hours later and transferred to cages with 8 wpf C57BL/6N males for mating. At 27 28 hours zygote 39 43 hours 2 cell 68 70 hours 8 cell and 92 94 hours blastocyst post hCG administration the female mice were euthanized by cervical dislocation. Embryos were flushed from the reproductive tract into HEPES buffered CZB medium then transferred to acidic Tyrode's solution for a few seconds to remove the zona pellucida followed by three washes in M2 medium. For zebrafish bulk zygote samples total RNA was extracted from 100 zebrafish zygotes using TRIzol™ Reagent and eluated in 100 μl of RNase free water. Then 10 μl and 5 μl samples were taken for 10 zygote and 5 zygote samples respectively and volumes were adjusted to 12 μl with nuclease free water. For zebrafish single zygote samples single zygote was manually picked and distributed into 12 μl of 1X Lysis buffer Takara. For the polyA selected RNA samples of mouse liver and embryonic stem cell WT and Mettl3 KO total RNA was extracted using TRIzol™ Reagent Thermo Fisher Scientific and PolyA+ RNA was selected twice by Dynabeads™ mRNA Purification Kit Thermo Fisher Scientific. For mouse embryonic stem cell samples the cells were sorted into 12 μl 1X Lysis buffer using BD FACSMelody™ Cell Sorter BD Biosciences. For mouse oocyte and embryo samples post three washes in M2 medium oocytes and embryos were manually picked and put into 12 μl 1X Lysis buffer. For picoMeRIP seq experiment NEBNext® rRNA Depletion Kit NEB was used to delete DNA and rRNA; RNA samples were sheared by sonication incubated with the anti m6A antibody from Millipore ABE572 and precipitated using ethanol. SMART Seq® Stranded Kit Takara Cat. # 634442 was used to prepare RNA sequencing library. Libraries were quantified with KAPA Library Quantification Kits Roche and the size distribution was checked using TapeStation D1000 ScreenTape Agilent Technologies.   developmental stage:Zygote|molecule subtype:rRNA free RNA GSM5599721 GSM5599721: ZF zygote IP 1cell Rep1; Danio rerio; RIP Seq GSM5599721   1 For mouse liver total RNA was extracted using TRIzol™ Reagent Thermo Fisher Scientific and PolyA+ RNA was selected twice by Dynabeads™ mRNA Purification Kit Thermo Fisher Scientific. For zebrafish zygote mouse MII oocyte and mouse blastocyst samples the single cells were manually picked and sorted into 12 μl 1X lysis buffer Takara; NEBNext® rRNA Depletion Kit NEB was used to delete DNA and rRNA; RNA samples were sonicated for 2 × 30 seconds incubated with the anti m6A antibody from Millipore ABE572 and precipitated using ethanol. SMART Seq® Stranded Kit Takara Cat. # 634442 was used to prepare RNA sequencing library. Libraries were quantified with KAPA Library Quantification Kits Roche and the size distribution was checked using TapeStation D1000 ScreenTape Agilent Technologies. GEO Accession:GSM5599721 RIP-Seq TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina NovaSeq 6000   SRP339082   loader:fastq load.py ZF_zygote_IP_1cell_Rep1_R1.fastq.gz ZF_zygote_IP_1cell_Rep1_R2.fastq.gz fastq fastq 1785761736.0 17507468.0 GSM5599721 r1 0:51 1:51 A:429081683;C:435813601;G:481376346;T:439419714;N:70392 51 51     429081683 435813601 481376346 439419714 70392 SRX12382353 SRS10353015 SRA1301707 GEO Room 2035, Palmer Commons, Department of Computational Medicine and Bioinformatics, University of Michigan 2 0.75314 0.75693 0.0543 0.06639 0.79482 0.79594 0.57979 0.56522 51 51 B B biological fallback assumption illumina novaseq_era 3prime poly_a nebnext bulk bulk bulk   United States 2021-09-28 Zygote Embryo Oocyte Reproductive System
66199 66199 SRR16096228 SRX12382352 SRS10353014 SRP339082 PRJNA766854 RNA m6A methylome from zebrafish zygotes mouse liver polyA selected RNAs mouse embryonic stem cells spike in RNA controls and single mouse oocytes and embryos GSE184893 Other N6 methyladenosine m6A is a prevalent RNA modification that has key roles in distinct biological processes including meiosis and early embryo development. Despite advances in methodology for m6A mapping the applicability of current methods is limited by the need for large amounts of input material. We develop a sensitive picogram scale m6A RNA immunoprecipitation and sequencing picoMeRIP seq method also suitable for single cell scMeRIP seq. We validate the down scaling of the method by profiling m6A from picogram amounts of mouse liver polyA selected RNA mouse embryonic stem cells and zebrafish zygotes. We provide proof of principle of m6A profiling in single mouse oocytes and preimplantation embryos. Our work opens a new avenue for studying m6A in single cells and scarce cell types in a transcriptome wide manner. Overall design: Transcriptome wide m6A maps were generated from zebrafish and mouse samples as below. 11 zebrafish zygote samples: 3 biological replicates for 10 zygotes 3 biological replicates for 5 zygotes and 5 biological replicates for single zygote. 6 mouse liver polyA selected RNA samples: 10 ng 1 ng and 100 pg; 2 biological replicates for each amount. 9 mouse embryonic stem cell R1 cell line from ATCC SCRC 1011 samples: 1000 cells 100 cells and 10 cells; 3 biological replicates for each amount. 3 spike in RNA control add 0.001 fmol Gaussia luciferase/GLuc with m6A modification and 0.001 fmol Cypridina luciferase/CLuc without xxx modification into the 12 ng polyA selected RNAs related samples: 12 ng polyA selected RNA from mouse liver; 6 ng polyA selected RNA from mouse liver and 6 ng polyA selected RNA from wild type/WT mouse embryonic stem cells from Geula S. et al. Science 2015; and 6 ng polyA selected RNA from mouse liver and 6 ng polyA selected RNA from Mettl3 knock out/KO mouse embryonic stem cells from Geula S. et al. Science 2015. 12 single mouse oocyte and embryo samples: oocytes at germinal vesicle GV and metaphase II MII stages and embryos at zygote 2 cell 8 cell and…   pubmed:37349523   ZF zygote IP 5cell Rep3 GSM5599720   tissue:ZF zygote IP 5cell|developmental stage:Zygote|molecule subtype:rRNA free RNA ZF zygote IP 5cell Rep3 Quality of raw reads was assessed using FastQC v0.11.8 and sequencing adapters were trimmed using Cutadapt v1.8.1 if clear adapter signal was detected at the three primeend of the reads. There is no adapter removal for zebrafish zygote samples GSM5599713 GSM5599714 GSM5599715 GSM5599716 GSM5599717 GSM5599718 GSM5599719 GSM5599720 GSM5599721 GSM5599722 GSM5599723 GSM5599724 GSM5599725 mouse polyA selected RNA samples GSM5599726 GSM5599727 GSM5599728 GSM5599729 GSM5599730 GSM5599731 GSM5599732 GSM5599733 and mouse MII oocyte Input sample GSM5599734. Trimmed reads were aligned to the reference genome mm10 for mouse danRer11 for zebrafish using HISAT2 v2.1.0 with the parameter “ 5 8 no mixed no discordant”. Multiply aligned reads were discarded as indicated by HISAT2. PCR duplicates were removed using Samtools v1.9. The read pairs that overlapped with ribosomal RNAs were removed using BEDTools v2.28.0. Genome coverage bigWig files bin size = 10 bp normalized by RPKM were generated by deepTools v3.2.0 bamCoverage with the parameter “ bs 10 normalizeUsing RPKM”. Genome build: mm10 for mouse danRer11 for zebrafish Supplementary files format and content: The bigWig files are generated by deepTools v3.2.0 bamCoverage with the bin size=10bp and normalized by RPKM. ZF zygote IP 5cell For mouse GV oocyte collection 8 wpf C57BL6/N females were injected with 5 units U of pregnant mare serum gonadotropin PMSG. post 48 hours ovaries were dissected and oocytes were isolated by puncturing the follicles. The procedure was carried out in M2 medium Sigma supplemented with 0.2 mM of the cyclic nucleotide phosphodiesterase inhibitor 3 Isobutyl 1 methylxanthine IBMX Sigma to prevent the oocytes from further progress to germinal vesicle breakdown GVBD. The cumulus cells were gently removed by pipetting and the oocytes were briefly exposed to acidic Tyrode's solution Sigma to remove the zona pellucida followed by three washes in M2 medium. For mouse MII oocyte collection 4 wpf 5 wpf C57BL6/N females were injected with 5 U of PMSG followed by 5 U of human chorionic gonadotropin hCG 45 hours later. The oviducts were dissected 20 22 hours later and transferred to a clean dish containing M2 medium. The oviduct ampulla was identified under a stereomicroscope to isolate MII oocytes containing the cumulus mass. The oocytes were treated with 0.3 mg/ml hyaluronidase dissolved in M2 medium to remove the cumulus cells and then exposed to acidic Tyrode's solution for a few seconds to remove the zona pellucida followed by three washes in M2 medium. For mouse early embryo collection 8 wpf C57BL/6N females were superovulated by hormone injection 5 U PMSG followed by 5 U hCG 45 hours later and transferred to cages with 8 wpf C57BL/6N males for mating. At 27 28 hours zygote 39 43 hours 2 cell 68 70 hours 8 cell and 92 94 hours blastocyst post hCG administration the female mice were euthanized by cervical dislocation. Embryos were flushed from the reproductive tract into HEPES buffered CZB medium then transferred to acidic Tyrode's solution for a few seconds to remove the zona pellucida followed by three washes in M2 medium. For zebrafish bulk zygote samples total RNA was extracted from 100 zebrafish zygotes using TRIzol™ Reagent and eluated in 100 μl of RNase free water. Then 10 μl and 5 μl samples were taken for 10 zygote and 5 zygote samples respectively and volumes were adjusted to 12 μl with nuclease free water. For zebrafish single zygote samples single zygote was manually picked and distributed into 12 μl of 1X Lysis buffer Takara. For the polyA selected RNA samples of mouse liver and embryonic stem cell WT and Mettl3 KO total RNA was extracted using TRIzol™ Reagent Thermo Fisher Scientific and PolyA+ RNA was selected twice by Dynabeads™ mRNA Purification Kit Thermo Fisher Scientific. For mouse embryonic stem cell samples the cells were sorted into 12 μl 1X Lysis buffer using BD FACSMelody™ Cell Sorter BD Biosciences. For mouse oocyte and embryo samples post three washes in M2 medium oocytes and embryos were manually picked and put into 12 μl 1X Lysis buffer. For picoMeRIP seq experiment NEBNext® rRNA Depletion Kit NEB was used to delete DNA and rRNA; RNA samples were sheared by sonication incubated with the anti m6A antibody from Millipore ABE572 and precipitated using ethanol. SMART Seq® Stranded Kit Takara Cat. # 634442 was used to prepare RNA sequencing library. Libraries were quantified with KAPA Library Quantification Kits Roche and the size distribution was checked using TapeStation D1000 ScreenTape Agilent Technologies.   developmental stage:Zygote|molecule subtype:rRNA free RNA GSM5599720 GSM5599720: ZF zygote IP 5cell Rep3; Danio rerio; RIP Seq GSM5599720   1 For mouse liver total RNA was extracted using TRIzol™ Reagent Thermo Fisher Scientific and PolyA+ RNA was selected twice by Dynabeads™ mRNA Purification Kit Thermo Fisher Scientific. For zebrafish zygote mouse MII oocyte and mouse blastocyst samples the single cells were manually picked and sorted into 12 μl 1X lysis buffer Takara; NEBNext® rRNA Depletion Kit NEB was used to delete DNA and rRNA; RNA samples were sonicated for 2 × 30 seconds incubated with the anti m6A antibody from Millipore ABE572 and precipitated using ethanol. SMART Seq® Stranded Kit Takara Cat. # 634442 was used to prepare RNA sequencing library. Libraries were quantified with KAPA Library Quantification Kits Roche and the size distribution was checked using TapeStation D1000 ScreenTape Agilent Technologies. GEO Accession:GSM5599720 RIP-Seq TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina NovaSeq 6000   SRP339082   loader:fastq load.py ZF_zygote_IP_5cell_Rep3_R1.fastq.gz ZF_zygote_IP_5cell_Rep3_R2.fastq.gz fastq fastq 2179659420.0 21369210.0 GSM5599720 r1 0:51 1:51 A:539092949;C:513857324;G:569620218;T:556940661;N:148268 51 51     539092949 513857324 569620218 556940661 148268 SRX12382352 SRS10353014 SRA1301707 GEO Room 2035, Palmer Commons, Department of Computational Medicine and Bioinformatics, University of Michigan 2 0.92028 0.90706 0.03967 0.05572 0.78431 0.78886 0.49727 0.48541 51 51 B B biological fallback assumption illumina novaseq_era 3prime poly_a nebnext bulk bulk bulk   United States 2021-09-28 Zygote Embryo Oocyte Reproductive System
66200 66200 SRR16096227 SRX12382351 SRS10353012 SRP339082 PRJNA766854 RNA m6A methylome from zebrafish zygotes mouse liver polyA selected RNAs mouse embryonic stem cells spike in RNA controls and single mouse oocytes and embryos GSE184893 Other N6 methyladenosine m6A is a prevalent RNA modification that has key roles in distinct biological processes including meiosis and early embryo development. Despite advances in methodology for m6A mapping the applicability of current methods is limited by the need for large amounts of input material. We develop a sensitive picogram scale m6A RNA immunoprecipitation and sequencing picoMeRIP seq method also suitable for single cell scMeRIP seq. We validate the down scaling of the method by profiling m6A from picogram amounts of mouse liver polyA selected RNA mouse embryonic stem cells and zebrafish zygotes. We provide proof of principle of m6A profiling in single mouse oocytes and preimplantation embryos. Our work opens a new avenue for studying m6A in single cells and scarce cell types in a transcriptome wide manner. Overall design: Transcriptome wide m6A maps were generated from zebrafish and mouse samples as below. 11 zebrafish zygote samples: 3 biological replicates for 10 zygotes 3 biological replicates for 5 zygotes and 5 biological replicates for single zygote. 6 mouse liver polyA selected RNA samples: 10 ng 1 ng and 100 pg; 2 biological replicates for each amount. 9 mouse embryonic stem cell R1 cell line from ATCC SCRC 1011 samples: 1000 cells 100 cells and 10 cells; 3 biological replicates for each amount. 3 spike in RNA control add 0.001 fmol Gaussia luciferase/GLuc with m6A modification and 0.001 fmol Cypridina luciferase/CLuc without xxx modification into the 12 ng polyA selected RNAs related samples: 12 ng polyA selected RNA from mouse liver; 6 ng polyA selected RNA from mouse liver and 6 ng polyA selected RNA from wild type/WT mouse embryonic stem cells from Geula S. et al. Science 2015; and 6 ng polyA selected RNA from mouse liver and 6 ng polyA selected RNA from Mettl3 knock out/KO mouse embryonic stem cells from Geula S. et al. Science 2015. 12 single mouse oocyte and embryo samples: oocytes at germinal vesicle GV and metaphase II MII stages and embryos at zygote 2 cell 8 cell and…   pubmed:37349523   ZF zygote IP 5cell Rep2 GSM5599719   tissue:ZF zygote IP 5cell|developmental stage:Zygote|molecule subtype:rRNA free RNA ZF zygote IP 5cell Rep2 Quality of raw reads was assessed using FastQC v0.11.8 and sequencing adapters were trimmed using Cutadapt v1.8.1 if clear adapter signal was detected at the three primeend of the reads. There is no adapter removal for zebrafish zygote samples GSM5599713 GSM5599714 GSM5599715 GSM5599716 GSM5599717 GSM5599718 GSM5599719 GSM5599720 GSM5599721 GSM5599722 GSM5599723 GSM5599724 GSM5599725 mouse polyA selected RNA samples GSM5599726 GSM5599727 GSM5599728 GSM5599729 GSM5599730 GSM5599731 GSM5599732 GSM5599733 and mouse MII oocyte Input sample GSM5599734. Trimmed reads were aligned to the reference genome mm10 for mouse danRer11 for zebrafish using HISAT2 v2.1.0 with the parameter “ 5 8 no mixed no discordant”. Multiply aligned reads were discarded as indicated by HISAT2. PCR duplicates were removed using Samtools v1.9. The read pairs that overlapped with ribosomal RNAs were removed using BEDTools v2.28.0. Genome coverage bigWig files bin size = 10 bp normalized by RPKM were generated by deepTools v3.2.0 bamCoverage with the parameter “ bs 10 normalizeUsing RPKM”. Genome build: mm10 for mouse danRer11 for zebrafish Supplementary files format and content: The bigWig files are generated by deepTools v3.2.0 bamCoverage with the bin size=10bp and normalized by RPKM. ZF zygote IP 5cell For mouse GV oocyte collection 8 wpf C57BL6/N females were injected with 5 units U of pregnant mare serum gonadotropin PMSG. post 48 hours ovaries were dissected and oocytes were isolated by puncturing the follicles. The procedure was carried out in M2 medium Sigma supplemented with 0.2 mM of the cyclic nucleotide phosphodiesterase inhibitor 3 Isobutyl 1 methylxanthine IBMX Sigma to prevent the oocytes from further progress to germinal vesicle breakdown GVBD. The cumulus cells were gently removed by pipetting and the oocytes were briefly exposed to acidic Tyrode's solution Sigma to remove the zona pellucida followed by three washes in M2 medium. For mouse MII oocyte collection 4 wpf 5 wpf C57BL6/N females were injected with 5 U of PMSG followed by 5 U of human chorionic gonadotropin hCG 45 hours later. The oviducts were dissected 20 22 hours later and transferred to a clean dish containing M2 medium. The oviduct ampulla was identified under a stereomicroscope to isolate MII oocytes containing the cumulus mass. The oocytes were treated with 0.3 mg/ml hyaluronidase dissolved in M2 medium to remove the cumulus cells and then exposed to acidic Tyrode's solution for a few seconds to remove the zona pellucida followed by three washes in M2 medium. For mouse early embryo collection 8 wpf C57BL/6N females were superovulated by hormone injection 5 U PMSG followed by 5 U hCG 45 hours later and transferred to cages with 8 wpf C57BL/6N males for mating. At 27 28 hours zygote 39 43 hours 2 cell 68 70 hours 8 cell and 92 94 hours blastocyst post hCG administration the female mice were euthanized by cervical dislocation. Embryos were flushed from the reproductive tract into HEPES buffered CZB medium then transferred to acidic Tyrode's solution for a few seconds to remove the zona pellucida followed by three washes in M2 medium. For zebrafish bulk zygote samples total RNA was extracted from 100 zebrafish zygotes using TRIzol™ Reagent and eluated in 100 μl of RNase free water. Then 10 μl and 5 μl samples were taken for 10 zygote and 5 zygote samples respectively and volumes were adjusted to 12 μl with nuclease free water. For zebrafish single zygote samples single zygote was manually picked and distributed into 12 μl of 1X Lysis buffer Takara. For the polyA selected RNA samples of mouse liver and embryonic stem cell WT and Mettl3 KO total RNA was extracted using TRIzol™ Reagent Thermo Fisher Scientific and PolyA+ RNA was selected twice by Dynabeads™ mRNA Purification Kit Thermo Fisher Scientific. For mouse embryonic stem cell samples the cells were sorted into 12 μl 1X Lysis buffer using BD FACSMelody™ Cell Sorter BD Biosciences. For mouse oocyte and embryo samples post three washes in M2 medium oocytes and embryos were manually picked and put into 12 μl 1X Lysis buffer. For picoMeRIP seq experiment NEBNext® rRNA Depletion Kit NEB was used to delete DNA and rRNA; RNA samples were sheared by sonication incubated with the anti m6A antibody from Millipore ABE572 and precipitated using ethanol. SMART Seq® Stranded Kit Takara Cat. # 634442 was used to prepare RNA sequencing library. Libraries were quantified with KAPA Library Quantification Kits Roche and the size distribution was checked using TapeStation D1000 ScreenTape Agilent Technologies.   developmental stage:Zygote|molecule subtype:rRNA free RNA GSM5599719 GSM5599719: ZF zygote IP 5cell Rep2; Danio rerio; RIP Seq GSM5599719   1 For mouse liver total RNA was extracted using TRIzol™ Reagent Thermo Fisher Scientific and PolyA+ RNA was selected twice by Dynabeads™ mRNA Purification Kit Thermo Fisher Scientific. For zebrafish zygote mouse MII oocyte and mouse blastocyst samples the single cells were manually picked and sorted into 12 μl 1X lysis buffer Takara; NEBNext® rRNA Depletion Kit NEB was used to delete DNA and rRNA; RNA samples were sonicated for 2 × 30 seconds incubated with the anti m6A antibody from Millipore ABE572 and precipitated using ethanol. SMART Seq® Stranded Kit Takara Cat. # 634442 was used to prepare RNA sequencing library. Libraries were quantified with KAPA Library Quantification Kits Roche and the size distribution was checked using TapeStation D1000 ScreenTape Agilent Technologies. GEO Accession:GSM5599719 RIP-Seq TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina NovaSeq 6000   SRP339082   loader:fastq load.py ZF_zygote_IP_5cell_Rep2_R1.fastq.gz ZF_zygote_IP_5cell_Rep2_R2.fastq.gz fastq fastq 2527184538.0 24776319.0 GSM5599719 r1 0:51 1:51 A:628307375;C:594702688;G:657847712;T:646151885;N:174878 51 51     628307375 594702688 657847712 646151885 174878 SRX12382351 SRS10353012 SRA1301707 GEO Room 2035, Palmer Commons, Department of Computational Medicine and Bioinformatics, University of Michigan 2 0.91811 0.90487 0.03919 0.05376 0.78315 0.78729 0.49414 0.48564 51 51 B B biological fallback assumption illumina novaseq_era 3prime poly_a nebnext bulk bulk bulk   United States 2021-09-28 Zygote Embryo Oocyte Reproductive System
66201 66201 SRR16096226 SRX12382350 SRS10353011 SRP339082 PRJNA766854 RNA m6A methylome from zebrafish zygotes mouse liver polyA selected RNAs mouse embryonic stem cells spike in RNA controls and single mouse oocytes and embryos GSE184893 Other N6 methyladenosine m6A is a prevalent RNA modification that has key roles in distinct biological processes including meiosis and early embryo development. Despite advances in methodology for m6A mapping the applicability of current methods is limited by the need for large amounts of input material. We develop a sensitive picogram scale m6A RNA immunoprecipitation and sequencing picoMeRIP seq method also suitable for single cell scMeRIP seq. We validate the down scaling of the method by profiling m6A from picogram amounts of mouse liver polyA selected RNA mouse embryonic stem cells and zebrafish zygotes. We provide proof of principle of m6A profiling in single mouse oocytes and preimplantation embryos. Our work opens a new avenue for studying m6A in single cells and scarce cell types in a transcriptome wide manner. Overall design: Transcriptome wide m6A maps were generated from zebrafish and mouse samples as below. 11 zebrafish zygote samples: 3 biological replicates for 10 zygotes 3 biological replicates for 5 zygotes and 5 biological replicates for single zygote. 6 mouse liver polyA selected RNA samples: 10 ng 1 ng and 100 pg; 2 biological replicates for each amount. 9 mouse embryonic stem cell R1 cell line from ATCC SCRC 1011 samples: 1000 cells 100 cells and 10 cells; 3 biological replicates for each amount. 3 spike in RNA control add 0.001 fmol Gaussia luciferase/GLuc with m6A modification and 0.001 fmol Cypridina luciferase/CLuc without xxx modification into the 12 ng polyA selected RNAs related samples: 12 ng polyA selected RNA from mouse liver; 6 ng polyA selected RNA from mouse liver and 6 ng polyA selected RNA from wild type/WT mouse embryonic stem cells from Geula S. et al. Science 2015; and 6 ng polyA selected RNA from mouse liver and 6 ng polyA selected RNA from Mettl3 knock out/KO mouse embryonic stem cells from Geula S. et al. Science 2015. 12 single mouse oocyte and embryo samples: oocytes at germinal vesicle GV and metaphase II MII stages and embryos at zygote 2 cell 8 cell and…   pubmed:37349523   ZF zygote IP 5cell Rep1 GSM5599718   tissue:ZF zygote IP 5cell|developmental stage:Zygote|molecule subtype:rRNA free RNA ZF zygote IP 5cell Rep1 Quality of raw reads was assessed using FastQC v0.11.8 and sequencing adapters were trimmed using Cutadapt v1.8.1 if clear adapter signal was detected at the three primeend of the reads. There is no adapter removal for zebrafish zygote samples GSM5599713 GSM5599714 GSM5599715 GSM5599716 GSM5599717 GSM5599718 GSM5599719 GSM5599720 GSM5599721 GSM5599722 GSM5599723 GSM5599724 GSM5599725 mouse polyA selected RNA samples GSM5599726 GSM5599727 GSM5599728 GSM5599729 GSM5599730 GSM5599731 GSM5599732 GSM5599733 and mouse MII oocyte Input sample GSM5599734. Trimmed reads were aligned to the reference genome mm10 for mouse danRer11 for zebrafish using HISAT2 v2.1.0 with the parameter “ 5 8 no mixed no discordant”. Multiply aligned reads were discarded as indicated by HISAT2. PCR duplicates were removed using Samtools v1.9. The read pairs that overlapped with ribosomal RNAs were removed using BEDTools v2.28.0. Genome coverage bigWig files bin size = 10 bp normalized by RPKM were generated by deepTools v3.2.0 bamCoverage with the parameter “ bs 10 normalizeUsing RPKM”. Genome build: mm10 for mouse danRer11 for zebrafish Supplementary files format and content: The bigWig files are generated by deepTools v3.2.0 bamCoverage with the bin size=10bp and normalized by RPKM. ZF zygote IP 5cell For mouse GV oocyte collection 8 wpf C57BL6/N females were injected with 5 units U of pregnant mare serum gonadotropin PMSG. post 48 hours ovaries were dissected and oocytes were isolated by puncturing the follicles. The procedure was carried out in M2 medium Sigma supplemented with 0.2 mM of the cyclic nucleotide phosphodiesterase inhibitor 3 Isobutyl 1 methylxanthine IBMX Sigma to prevent the oocytes from further progress to germinal vesicle breakdown GVBD. The cumulus cells were gently removed by pipetting and the oocytes were briefly exposed to acidic Tyrode's solution Sigma to remove the zona pellucida followed by three washes in M2 medium. For mouse MII oocyte collection 4 wpf 5 wpf C57BL6/N females were injected with 5 U of PMSG followed by 5 U of human chorionic gonadotropin hCG 45 hours later. The oviducts were dissected 20 22 hours later and transferred to a clean dish containing M2 medium. The oviduct ampulla was identified under a stereomicroscope to isolate MII oocytes containing the cumulus mass. The oocytes were treated with 0.3 mg/ml hyaluronidase dissolved in M2 medium to remove the cumulus cells and then exposed to acidic Tyrode's solution for a few seconds to remove the zona pellucida followed by three washes in M2 medium. For mouse early embryo collection 8 wpf C57BL/6N females were superovulated by hormone injection 5 U PMSG followed by 5 U hCG 45 hours later and transferred to cages with 8 wpf C57BL/6N males for mating. At 27 28 hours zygote 39 43 hours 2 cell 68 70 hours 8 cell and 92 94 hours blastocyst post hCG administration the female mice were euthanized by cervical dislocation. Embryos were flushed from the reproductive tract into HEPES buffered CZB medium then transferred to acidic Tyrode's solution for a few seconds to remove the zona pellucida followed by three washes in M2 medium. For zebrafish bulk zygote samples total RNA was extracted from 100 zebrafish zygotes using TRIzol™ Reagent and eluated in 100 μl of RNase free water. Then 10 μl and 5 μl samples were taken for 10 zygote and 5 zygote samples respectively and volumes were adjusted to 12 μl with nuclease free water. For zebrafish single zygote samples single zygote was manually picked and distributed into 12 μl of 1X Lysis buffer Takara. For the polyA selected RNA samples of mouse liver and embryonic stem cell WT and Mettl3 KO total RNA was extracted using TRIzol™ Reagent Thermo Fisher Scientific and PolyA+ RNA was selected twice by Dynabeads™ mRNA Purification Kit Thermo Fisher Scientific. For mouse embryonic stem cell samples the cells were sorted into 12 μl 1X Lysis buffer using BD FACSMelody™ Cell Sorter BD Biosciences. For mouse oocyte and embryo samples post three washes in M2 medium oocytes and embryos were manually picked and put into 12 μl 1X Lysis buffer. For picoMeRIP seq experiment NEBNext® rRNA Depletion Kit NEB was used to delete DNA and rRNA; RNA samples were sheared by sonication incubated with the anti m6A antibody from Millipore ABE572 and precipitated using ethanol. SMART Seq® Stranded Kit Takara Cat. # 634442 was used to prepare RNA sequencing library. Libraries were quantified with KAPA Library Quantification Kits Roche and the size distribution was checked using TapeStation D1000 ScreenTape Agilent Technologies.   developmental stage:Zygote|molecule subtype:rRNA free RNA GSM5599718 GSM5599718: ZF zygote IP 5cell Rep1; Danio rerio; RIP Seq GSM5599718   1 For mouse liver total RNA was extracted using TRIzol™ Reagent Thermo Fisher Scientific and PolyA+ RNA was selected twice by Dynabeads™ mRNA Purification Kit Thermo Fisher Scientific. For zebrafish zygote mouse MII oocyte and mouse blastocyst samples the single cells were manually picked and sorted into 12 μl 1X lysis buffer Takara; NEBNext® rRNA Depletion Kit NEB was used to delete DNA and rRNA; RNA samples were sonicated for 2 × 30 seconds incubated with the anti m6A antibody from Millipore ABE572 and precipitated using ethanol. SMART Seq® Stranded Kit Takara Cat. # 634442 was used to prepare RNA sequencing library. Libraries were quantified with KAPA Library Quantification Kits Roche and the size distribution was checked using TapeStation D1000 ScreenTape Agilent Technologies. GEO Accession:GSM5599718 RIP-Seq TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina NovaSeq 6000   SRP339082   loader:fastq load.py ZF_zygote_IP_5cell_Rep1_R1.fastq.gz ZF_zygote_IP_5cell_Rep1_R2.fastq.gz fastq fastq 3481342518.0 34130809.0 GSM5599718 r1 0:51 1:51 A:865680250;C:823420389;G:907146965;T:884854199;N:240715 51 51     865680250 823420389 907146965 884854199 240715 SRX12382350 SRS10353011 SRA1301707 GEO Room 2035, Palmer Commons, Department of Computational Medicine and Bioinformatics, University of Michigan 2 0.91893 0.90939 0.04544 0.06005 0.78025 0.78348 0.50569 0.49155 51 51 B B biological fallback assumption illumina novaseq_era 3prime poly_a nebnext bulk bulk bulk   United States 2021-09-28 Zygote Embryo Oocyte Reproductive System
66202 66202 SRR16096225 SRX12382349 SRS10353013 SRP339082 PRJNA766854 RNA m6A methylome from zebrafish zygotes mouse liver polyA selected RNAs mouse embryonic stem cells spike in RNA controls and single mouse oocytes and embryos GSE184893 Other N6 methyladenosine m6A is a prevalent RNA modification that has key roles in distinct biological processes including meiosis and early embryo development. Despite advances in methodology for m6A mapping the applicability of current methods is limited by the need for large amounts of input material. We develop a sensitive picogram scale m6A RNA immunoprecipitation and sequencing picoMeRIP seq method also suitable for single cell scMeRIP seq. We validate the down scaling of the method by profiling m6A from picogram amounts of mouse liver polyA selected RNA mouse embryonic stem cells and zebrafish zygotes. We provide proof of principle of m6A profiling in single mouse oocytes and preimplantation embryos. Our work opens a new avenue for studying m6A in single cells and scarce cell types in a transcriptome wide manner. Overall design: Transcriptome wide m6A maps were generated from zebrafish and mouse samples as below. 11 zebrafish zygote samples: 3 biological replicates for 10 zygotes 3 biological replicates for 5 zygotes and 5 biological replicates for single zygote. 6 mouse liver polyA selected RNA samples: 10 ng 1 ng and 100 pg; 2 biological replicates for each amount. 9 mouse embryonic stem cell R1 cell line from ATCC SCRC 1011 samples: 1000 cells 100 cells and 10 cells; 3 biological replicates for each amount. 3 spike in RNA control add 0.001 fmol Gaussia luciferase/GLuc with m6A modification and 0.001 fmol Cypridina luciferase/CLuc without xxx modification into the 12 ng polyA selected RNAs related samples: 12 ng polyA selected RNA from mouse liver; 6 ng polyA selected RNA from mouse liver and 6 ng polyA selected RNA from wild type/WT mouse embryonic stem cells from Geula S. et al. Science 2015; and 6 ng polyA selected RNA from mouse liver and 6 ng polyA selected RNA from Mettl3 knock out/KO mouse embryonic stem cells from Geula S. et al. Science 2015. 12 single mouse oocyte and embryo samples: oocytes at germinal vesicle GV and metaphase II MII stages and embryos at zygote 2 cell 8 cell and…   pubmed:37349523   ZF zygote IP 10cell Rep3 GSM5599717   tissue:ZF zygote IP 10cell|developmental stage:Zygote|molecule subtype:rRNA free RNA ZF zygote IP 10cell Rep3 Quality of raw reads was assessed using FastQC v0.11.8 and sequencing adapters were trimmed using Cutadapt v1.8.1 if clear adapter signal was detected at the three primeend of the reads. There is no adapter removal for zebrafish zygote samples GSM5599713 GSM5599714 GSM5599715 GSM5599716 GSM5599717 GSM5599718 GSM5599719 GSM5599720 GSM5599721 GSM5599722 GSM5599723 GSM5599724 GSM5599725 mouse polyA selected RNA samples GSM5599726 GSM5599727 GSM5599728 GSM5599729 GSM5599730 GSM5599731 GSM5599732 GSM5599733 and mouse MII oocyte Input sample GSM5599734. Trimmed reads were aligned to the reference genome mm10 for mouse danRer11 for zebrafish using HISAT2 v2.1.0 with the parameter “ 5 8 no mixed no discordant”. Multiply aligned reads were discarded as indicated by HISAT2. PCR duplicates were removed using Samtools v1.9. The read pairs that overlapped with ribosomal RNAs were removed using BEDTools v2.28.0. Genome coverage bigWig files bin size = 10 bp normalized by RPKM were generated by deepTools v3.2.0 bamCoverage with the parameter “ bs 10 normalizeUsing RPKM”. Genome build: mm10 for mouse danRer11 for zebrafish Supplementary files format and content: The bigWig files are generated by deepTools v3.2.0 bamCoverage with the bin size=10bp and normalized by RPKM. ZF zygote IP 10cell For mouse GV oocyte collection 8 wpf C57BL6/N females were injected with 5 units U of pregnant mare serum gonadotropin PMSG. post 48 hours ovaries were dissected and oocytes were isolated by puncturing the follicles. The procedure was carried out in M2 medium Sigma supplemented with 0.2 mM of the cyclic nucleotide phosphodiesterase inhibitor 3 Isobutyl 1 methylxanthine IBMX Sigma to prevent the oocytes from further progress to germinal vesicle breakdown GVBD. The cumulus cells were gently removed by pipetting and the oocytes were briefly exposed to acidic Tyrode's solution Sigma to remove the zona pellucida followed by three washes in M2 medium. For mouse MII oocyte collection 4 wpf 5 wpf C57BL6/N females were injected with 5 U of PMSG followed by 5 U of human chorionic gonadotropin hCG 45 hours later. The oviducts were dissected 20 22 hours later and transferred to a clean dish containing M2 medium. The oviduct ampulla was identified under a stereomicroscope to isolate MII oocytes containing the cumulus mass. The oocytes were treated with 0.3 mg/ml hyaluronidase dissolved in M2 medium to remove the cumulus cells and then exposed to acidic Tyrode's solution for a few seconds to remove the zona pellucida followed by three washes in M2 medium. For mouse early embryo collection 8 wpf C57BL/6N females were superovulated by hormone injection 5 U PMSG followed by 5 U hCG 45 hours later and transferred to cages with 8 wpf C57BL/6N males for mating. At 27 28 hours zygote 39 43 hours 2 cell 68 70 hours 8 cell and 92 94 hours blastocyst post hCG administration the female mice were euthanized by cervical dislocation. Embryos were flushed from the reproductive tract into HEPES buffered CZB medium then transferred to acidic Tyrode's solution for a few seconds to remove the zona pellucida followed by three washes in M2 medium. For zebrafish bulk zygote samples total RNA was extracted from 100 zebrafish zygotes using TRIzol™ Reagent and eluated in 100 μl of RNase free water. Then 10 μl and 5 μl samples were taken for 10 zygote and 5 zygote samples respectively and volumes were adjusted to 12 μl with nuclease free water. For zebrafish single zygote samples single zygote was manually picked and distributed into 12 μl of 1X Lysis buffer Takara. For the polyA selected RNA samples of mouse liver and embryonic stem cell WT and Mettl3 KO total RNA was extracted using TRIzol™ Reagent Thermo Fisher Scientific and PolyA+ RNA was selected twice by Dynabeads™ mRNA Purification Kit Thermo Fisher Scientific. For mouse embryonic stem cell samples the cells were sorted into 12 μl 1X Lysis buffer using BD FACSMelody™ Cell Sorter BD Biosciences. For mouse oocyte and embryo samples post three washes in M2 medium oocytes and embryos were manually picked and put into 12 μl 1X Lysis buffer. For picoMeRIP seq experiment NEBNext® rRNA Depletion Kit NEB was used to delete DNA and rRNA; RNA samples were sheared by sonication incubated with the anti m6A antibody from Millipore ABE572 and precipitated using ethanol. SMART Seq® Stranded Kit Takara Cat. # 634442 was used to prepare RNA sequencing library. Libraries were quantified with KAPA Library Quantification Kits Roche and the size distribution was checked using TapeStation D1000 ScreenTape Agilent Technologies.   developmental stage:Zygote|molecule subtype:rRNA free RNA GSM5599717 GSM5599717: ZF zygote IP 10cell Rep3; Danio rerio; RIP Seq GSM5599717   1 For mouse liver total RNA was extracted using TRIzol™ Reagent Thermo Fisher Scientific and PolyA+ RNA was selected twice by Dynabeads™ mRNA Purification Kit Thermo Fisher Scientific. For zebrafish zygote mouse MII oocyte and mouse blastocyst samples the single cells were manually picked and sorted into 12 μl 1X lysis buffer Takara; NEBNext® rRNA Depletion Kit NEB was used to delete DNA and rRNA; RNA samples were sonicated for 2 × 30 seconds incubated with the anti m6A antibody from Millipore ABE572 and precipitated using ethanol. SMART Seq® Stranded Kit Takara Cat. # 634442 was used to prepare RNA sequencing library. Libraries were quantified with KAPA Library Quantification Kits Roche and the size distribution was checked using TapeStation D1000 ScreenTape Agilent Technologies. GEO Accession:GSM5599717 RIP-Seq TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina NovaSeq 6000   SRP339082   loader:fastq load.py ZF_zygote_IP_10cell_Rep3_R1.fastq.gz ZF_zygote_IP_10cell_Rep3_R2.fastq.gz fastq fastq 2172362850.0 21297675.0 GSM5599717 r1 0:51 1:51 A:536043605;C:513512883;G:568368414;T:554284996;N:152952 51 51     536043605 513512883 568368414 554284996 152952 SRX12382349 SRS10353013 SRA1301707 GEO Room 2035, Palmer Commons, Department of Computational Medicine and Bioinformatics, University of Michigan 2 0.91859 0.90522 0.03736 0.05214 0.78715 0.79129 0.49736 0.48681 51 51 B B biological fallback assumption illumina novaseq_era 3prime poly_a nebnext bulk bulk bulk   United States 2021-09-28 Zygote Embryo Oocyte Reproductive System
66203 66203 SRR16096224 SRX12382348 SRS10353010 SRP339082 PRJNA766854 RNA m6A methylome from zebrafish zygotes mouse liver polyA selected RNAs mouse embryonic stem cells spike in RNA controls and single mouse oocytes and embryos GSE184893 Other N6 methyladenosine m6A is a prevalent RNA modification that has key roles in distinct biological processes including meiosis and early embryo development. Despite advances in methodology for m6A mapping the applicability of current methods is limited by the need for large amounts of input material. We develop a sensitive picogram scale m6A RNA immunoprecipitation and sequencing picoMeRIP seq method also suitable for single cell scMeRIP seq. We validate the down scaling of the method by profiling m6A from picogram amounts of mouse liver polyA selected RNA mouse embryonic stem cells and zebrafish zygotes. We provide proof of principle of m6A profiling in single mouse oocytes and preimplantation embryos. Our work opens a new avenue for studying m6A in single cells and scarce cell types in a transcriptome wide manner. Overall design: Transcriptome wide m6A maps were generated from zebrafish and mouse samples as below. 11 zebrafish zygote samples: 3 biological replicates for 10 zygotes 3 biological replicates for 5 zygotes and 5 biological replicates for single zygote. 6 mouse liver polyA selected RNA samples: 10 ng 1 ng and 100 pg; 2 biological replicates for each amount. 9 mouse embryonic stem cell R1 cell line from ATCC SCRC 1011 samples: 1000 cells 100 cells and 10 cells; 3 biological replicates for each amount. 3 spike in RNA control add 0.001 fmol Gaussia luciferase/GLuc with m6A modification and 0.001 fmol Cypridina luciferase/CLuc without xxx modification into the 12 ng polyA selected RNAs related samples: 12 ng polyA selected RNA from mouse liver; 6 ng polyA selected RNA from mouse liver and 6 ng polyA selected RNA from wild type/WT mouse embryonic stem cells from Geula S. et al. Science 2015; and 6 ng polyA selected RNA from mouse liver and 6 ng polyA selected RNA from Mettl3 knock out/KO mouse embryonic stem cells from Geula S. et al. Science 2015. 12 single mouse oocyte and embryo samples: oocytes at germinal vesicle GV and metaphase II MII stages and embryos at zygote 2 cell 8 cell and…   pubmed:37349523   ZF zygote IP 10cell Rep2 GSM5599716   tissue:ZF zygote IP 10cell|developmental stage:Zygote|molecule subtype:rRNA free RNA ZF zygote IP 10cell Rep2 Quality of raw reads was assessed using FastQC v0.11.8 and sequencing adapters were trimmed using Cutadapt v1.8.1 if clear adapter signal was detected at the three primeend of the reads. There is no adapter removal for zebrafish zygote samples GSM5599713 GSM5599714 GSM5599715 GSM5599716 GSM5599717 GSM5599718 GSM5599719 GSM5599720 GSM5599721 GSM5599722 GSM5599723 GSM5599724 GSM5599725 mouse polyA selected RNA samples GSM5599726 GSM5599727 GSM5599728 GSM5599729 GSM5599730 GSM5599731 GSM5599732 GSM5599733 and mouse MII oocyte Input sample GSM5599734. Trimmed reads were aligned to the reference genome mm10 for mouse danRer11 for zebrafish using HISAT2 v2.1.0 with the parameter “ 5 8 no mixed no discordant”. Multiply aligned reads were discarded as indicated by HISAT2. PCR duplicates were removed using Samtools v1.9. The read pairs that overlapped with ribosomal RNAs were removed using BEDTools v2.28.0. Genome coverage bigWig files bin size = 10 bp normalized by RPKM were generated by deepTools v3.2.0 bamCoverage with the parameter “ bs 10 normalizeUsing RPKM”. Genome build: mm10 for mouse danRer11 for zebrafish Supplementary files format and content: The bigWig files are generated by deepTools v3.2.0 bamCoverage with the bin size=10bp and normalized by RPKM. ZF zygote IP 10cell For mouse GV oocyte collection 8 wpf C57BL6/N females were injected with 5 units U of pregnant mare serum gonadotropin PMSG. post 48 hours ovaries were dissected and oocytes were isolated by puncturing the follicles. The procedure was carried out in M2 medium Sigma supplemented with 0.2 mM of the cyclic nucleotide phosphodiesterase inhibitor 3 Isobutyl 1 methylxanthine IBMX Sigma to prevent the oocytes from further progress to germinal vesicle breakdown GVBD. The cumulus cells were gently removed by pipetting and the oocytes were briefly exposed to acidic Tyrode's solution Sigma to remove the zona pellucida followed by three washes in M2 medium. For mouse MII oocyte collection 4 wpf 5 wpf C57BL6/N females were injected with 5 U of PMSG followed by 5 U of human chorionic gonadotropin hCG 45 hours later. The oviducts were dissected 20 22 hours later and transferred to a clean dish containing M2 medium. The oviduct ampulla was identified under a stereomicroscope to isolate MII oocytes containing the cumulus mass. The oocytes were treated with 0.3 mg/ml hyaluronidase dissolved in M2 medium to remove the cumulus cells and then exposed to acidic Tyrode's solution for a few seconds to remove the zona pellucida followed by three washes in M2 medium. For mouse early embryo collection 8 wpf C57BL/6N females were superovulated by hormone injection 5 U PMSG followed by 5 U hCG 45 hours later and transferred to cages with 8 wpf C57BL/6N males for mating. At 27 28 hours zygote 39 43 hours 2 cell 68 70 hours 8 cell and 92 94 hours blastocyst post hCG administration the female mice were euthanized by cervical dislocation. Embryos were flushed from the reproductive tract into HEPES buffered CZB medium then transferred to acidic Tyrode's solution for a few seconds to remove the zona pellucida followed by three washes in M2 medium. For zebrafish bulk zygote samples total RNA was extracted from 100 zebrafish zygotes using TRIzol™ Reagent and eluated in 100 μl of RNase free water. Then 10 μl and 5 μl samples were taken for 10 zygote and 5 zygote samples respectively and volumes were adjusted to 12 μl with nuclease free water. For zebrafish single zygote samples single zygote was manually picked and distributed into 12 μl of 1X Lysis buffer Takara. For the polyA selected RNA samples of mouse liver and embryonic stem cell WT and Mettl3 KO total RNA was extracted using TRIzol™ Reagent Thermo Fisher Scientific and PolyA+ RNA was selected twice by Dynabeads™ mRNA Purification Kit Thermo Fisher Scientific. For mouse embryonic stem cell samples the cells were sorted into 12 μl 1X Lysis buffer using BD FACSMelody™ Cell Sorter BD Biosciences. For mouse oocyte and embryo samples post three washes in M2 medium oocytes and embryos were manually picked and put into 12 μl 1X Lysis buffer. For picoMeRIP seq experiment NEBNext® rRNA Depletion Kit NEB was used to delete DNA and rRNA; RNA samples were sheared by sonication incubated with the anti m6A antibody from Millipore ABE572 and precipitated using ethanol. SMART Seq® Stranded Kit Takara Cat. # 634442 was used to prepare RNA sequencing library. Libraries were quantified with KAPA Library Quantification Kits Roche and the size distribution was checked using TapeStation D1000 ScreenTape Agilent Technologies.   developmental stage:Zygote|molecule subtype:rRNA free RNA GSM5599716 GSM5599716: ZF zygote IP 10cell Rep2; Danio rerio; RIP Seq GSM5599716   1 For mouse liver total RNA was extracted using TRIzol™ Reagent Thermo Fisher Scientific and PolyA+ RNA was selected twice by Dynabeads™ mRNA Purification Kit Thermo Fisher Scientific. For zebrafish zygote mouse MII oocyte and mouse blastocyst samples the single cells were manually picked and sorted into 12 μl 1X lysis buffer Takara; NEBNext® rRNA Depletion Kit NEB was used to delete DNA and rRNA; RNA samples were sonicated for 2 × 30 seconds incubated with the anti m6A antibody from Millipore ABE572 and precipitated using ethanol. SMART Seq® Stranded Kit Takara Cat. # 634442 was used to prepare RNA sequencing library. Libraries were quantified with KAPA Library Quantification Kits Roche and the size distribution was checked using TapeStation D1000 ScreenTape Agilent Technologies. GEO Accession:GSM5599716 RIP-Seq TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina NovaSeq 6000   SRP339082   loader:fastq load.py ZF_zygote_IP_10cell_Rep2_R1.fastq.gz ZF_zygote_IP_10cell_Rep2_R2.fastq.gz fastq fastq 1840839492.0 18047446.0 GSM5599716 r1 0:51 1:51 A:454385290;C:437082102;G:482351138;T:466893642;N:127320 51 51     454385290 437082102 482351138 466893642 127320 SRX12382348 SRS10353010 SRA1301707 GEO Room 2035, Palmer Commons, Department of Computational Medicine and Bioinformatics, University of Michigan 2 0.92217 0.90956 0.04012 0.05483 0.78819 0.79318 0.49756 0.48256 51 51 B B biological fallback assumption illumina novaseq_era 3prime poly_a nebnext bulk bulk bulk   United States 2021-09-28 Zygote Embryo Oocyte Reproductive System
66204 66204 SRR16096223 SRX12382347 SRS10353009 SRP339082 PRJNA766854 RNA m6A methylome from zebrafish zygotes mouse liver polyA selected RNAs mouse embryonic stem cells spike in RNA controls and single mouse oocytes and embryos GSE184893 Other N6 methyladenosine m6A is a prevalent RNA modification that has key roles in distinct biological processes including meiosis and early embryo development. Despite advances in methodology for m6A mapping the applicability of current methods is limited by the need for large amounts of input material. We develop a sensitive picogram scale m6A RNA immunoprecipitation and sequencing picoMeRIP seq method also suitable for single cell scMeRIP seq. We validate the down scaling of the method by profiling m6A from picogram amounts of mouse liver polyA selected RNA mouse embryonic stem cells and zebrafish zygotes. We provide proof of principle of m6A profiling in single mouse oocytes and preimplantation embryos. Our work opens a new avenue for studying m6A in single cells and scarce cell types in a transcriptome wide manner. Overall design: Transcriptome wide m6A maps were generated from zebrafish and mouse samples as below. 11 zebrafish zygote samples: 3 biological replicates for 10 zygotes 3 biological replicates for 5 zygotes and 5 biological replicates for single zygote. 6 mouse liver polyA selected RNA samples: 10 ng 1 ng and 100 pg; 2 biological replicates for each amount. 9 mouse embryonic stem cell R1 cell line from ATCC SCRC 1011 samples: 1000 cells 100 cells and 10 cells; 3 biological replicates for each amount. 3 spike in RNA control add 0.001 fmol Gaussia luciferase/GLuc with m6A modification and 0.001 fmol Cypridina luciferase/CLuc without xxx modification into the 12 ng polyA selected RNAs related samples: 12 ng polyA selected RNA from mouse liver; 6 ng polyA selected RNA from mouse liver and 6 ng polyA selected RNA from wild type/WT mouse embryonic stem cells from Geula S. et al. Science 2015; and 6 ng polyA selected RNA from mouse liver and 6 ng polyA selected RNA from Mettl3 knock out/KO mouse embryonic stem cells from Geula S. et al. Science 2015. 12 single mouse oocyte and embryo samples: oocytes at germinal vesicle GV and metaphase II MII stages and embryos at zygote 2 cell 8 cell and…   pubmed:37349523   ZF zygote IP 10cell Rep1 GSM5599715   tissue:ZF zygote IP 10cell|developmental stage:Zygote|molecule subtype:rRNA free RNA ZF zygote IP 10cell Rep1 Quality of raw reads was assessed using FastQC v0.11.8 and sequencing adapters were trimmed using Cutadapt v1.8.1 if clear adapter signal was detected at the three primeend of the reads. There is no adapter removal for zebrafish zygote samples GSM5599713 GSM5599714 GSM5599715 GSM5599716 GSM5599717 GSM5599718 GSM5599719 GSM5599720 GSM5599721 GSM5599722 GSM5599723 GSM5599724 GSM5599725 mouse polyA selected RNA samples GSM5599726 GSM5599727 GSM5599728 GSM5599729 GSM5599730 GSM5599731 GSM5599732 GSM5599733 and mouse MII oocyte Input sample GSM5599734. Trimmed reads were aligned to the reference genome mm10 for mouse danRer11 for zebrafish using HISAT2 v2.1.0 with the parameter “ 5 8 no mixed no discordant”. Multiply aligned reads were discarded as indicated by HISAT2. PCR duplicates were removed using Samtools v1.9. The read pairs that overlapped with ribosomal RNAs were removed using BEDTools v2.28.0. Genome coverage bigWig files bin size = 10 bp normalized by RPKM were generated by deepTools v3.2.0 bamCoverage with the parameter “ bs 10 normalizeUsing RPKM”. Genome build: mm10 for mouse danRer11 for zebrafish Supplementary files format and content: The bigWig files are generated by deepTools v3.2.0 bamCoverage with the bin size=10bp and normalized by RPKM. ZF zygote IP 10cell For mouse GV oocyte collection 8 wpf C57BL6/N females were injected with 5 units U of pregnant mare serum gonadotropin PMSG. post 48 hours ovaries were dissected and oocytes were isolated by puncturing the follicles. The procedure was carried out in M2 medium Sigma supplemented with 0.2 mM of the cyclic nucleotide phosphodiesterase inhibitor 3 Isobutyl 1 methylxanthine IBMX Sigma to prevent the oocytes from further progress to germinal vesicle breakdown GVBD. The cumulus cells were gently removed by pipetting and the oocytes were briefly exposed to acidic Tyrode's solution Sigma to remove the zona pellucida followed by three washes in M2 medium. For mouse MII oocyte collection 4 wpf 5 wpf C57BL6/N females were injected with 5 U of PMSG followed by 5 U of human chorionic gonadotropin hCG 45 hours later. The oviducts were dissected 20 22 hours later and transferred to a clean dish containing M2 medium. The oviduct ampulla was identified under a stereomicroscope to isolate MII oocytes containing the cumulus mass. The oocytes were treated with 0.3 mg/ml hyaluronidase dissolved in M2 medium to remove the cumulus cells and then exposed to acidic Tyrode's solution for a few seconds to remove the zona pellucida followed by three washes in M2 medium. For mouse early embryo collection 8 wpf C57BL/6N females were superovulated by hormone injection 5 U PMSG followed by 5 U hCG 45 hours later and transferred to cages with 8 wpf C57BL/6N males for mating. At 27 28 hours zygote 39 43 hours 2 cell 68 70 hours 8 cell and 92 94 hours blastocyst post hCG administration the female mice were euthanized by cervical dislocation. Embryos were flushed from the reproductive tract into HEPES buffered CZB medium then transferred to acidic Tyrode's solution for a few seconds to remove the zona pellucida followed by three washes in M2 medium. For zebrafish bulk zygote samples total RNA was extracted from 100 zebrafish zygotes using TRIzol™ Reagent and eluated in 100 μl of RNase free water. Then 10 μl and 5 μl samples were taken for 10 zygote and 5 zygote samples respectively and volumes were adjusted to 12 μl with nuclease free water. For zebrafish single zygote samples single zygote was manually picked and distributed into 12 μl of 1X Lysis buffer Takara. For the polyA selected RNA samples of mouse liver and embryonic stem cell WT and Mettl3 KO total RNA was extracted using TRIzol™ Reagent Thermo Fisher Scientific and PolyA+ RNA was selected twice by Dynabeads™ mRNA Purification Kit Thermo Fisher Scientific. For mouse embryonic stem cell samples the cells were sorted into 12 μl 1X Lysis buffer using BD FACSMelody™ Cell Sorter BD Biosciences. For mouse oocyte and embryo samples post three washes in M2 medium oocytes and embryos were manually picked and put into 12 μl 1X Lysis buffer. For picoMeRIP seq experiment NEBNext® rRNA Depletion Kit NEB was used to delete DNA and rRNA; RNA samples were sheared by sonication incubated with the anti m6A antibody from Millipore ABE572 and precipitated using ethanol. SMART Seq® Stranded Kit Takara Cat. # 634442 was used to prepare RNA sequencing library. Libraries were quantified with KAPA Library Quantification Kits Roche and the size distribution was checked using TapeStation D1000 ScreenTape Agilent Technologies.   developmental stage:Zygote|molecule subtype:rRNA free RNA GSM5599715 GSM5599715: ZF zygote IP 10cell Rep1; Danio rerio; RIP Seq GSM5599715   1 For mouse liver total RNA was extracted using TRIzol™ Reagent Thermo Fisher Scientific and PolyA+ RNA was selected twice by Dynabeads™ mRNA Purification Kit Thermo Fisher Scientific. For zebrafish zygote mouse MII oocyte and mouse blastocyst samples the single cells were manually picked and sorted into 12 μl 1X lysis buffer Takara; NEBNext® rRNA Depletion Kit NEB was used to delete DNA and rRNA; RNA samples were sonicated for 2 × 30 seconds incubated with the anti m6A antibody from Millipore ABE572 and precipitated using ethanol. SMART Seq® Stranded Kit Takara Cat. # 634442 was used to prepare RNA sequencing library. Libraries were quantified with KAPA Library Quantification Kits Roche and the size distribution was checked using TapeStation D1000 ScreenTape Agilent Technologies. GEO Accession:GSM5599715 RIP-Seq TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina NovaSeq 6000   SRP339082   loader:fastq load.py ZF_zygote_IP_10cell_Rep1_R1.fastq.gz ZF_zygote_IP_10cell_Rep1_R2.fastq.gz fastq fastq 2400553680.0 23534840.0 GSM5599715 r1 0:51 1:51 A:594878203;C:567573525;G:625488388;T:612448386;N:165178 51 51     594878203 567573525 625488388 612448386 165178 SRX12382347 SRS10353009 SRA1301707 GEO Room 2035, Palmer Commons, Department of Computational Medicine and Bioinformatics, University of Michigan 2 0.91641 0.908 0.03979 0.05394 0.78455 0.7892 0.48862 0.47478 51 51 B B biological fallback assumption illumina novaseq_era 3prime poly_a nebnext bulk bulk bulk   United States 2021-09-28 Zygote Embryo Oocyte Reproductive System
66205 66205 SRR16096222 SRX12382346 SRS10353008 SRP339082 PRJNA766854 RNA m6A methylome from zebrafish zygotes mouse liver polyA selected RNAs mouse embryonic stem cells spike in RNA controls and single mouse oocytes and embryos GSE184893 Other N6 methyladenosine m6A is a prevalent RNA modification that has key roles in distinct biological processes including meiosis and early embryo development. Despite advances in methodology for m6A mapping the applicability of current methods is limited by the need for large amounts of input material. We develop a sensitive picogram scale m6A RNA immunoprecipitation and sequencing picoMeRIP seq method also suitable for single cell scMeRIP seq. We validate the down scaling of the method by profiling m6A from picogram amounts of mouse liver polyA selected RNA mouse embryonic stem cells and zebrafish zygotes. We provide proof of principle of m6A profiling in single mouse oocytes and preimplantation embryos. Our work opens a new avenue for studying m6A in single cells and scarce cell types in a transcriptome wide manner. Overall design: Transcriptome wide m6A maps were generated from zebrafish and mouse samples as below. 11 zebrafish zygote samples: 3 biological replicates for 10 zygotes 3 biological replicates for 5 zygotes and 5 biological replicates for single zygote. 6 mouse liver polyA selected RNA samples: 10 ng 1 ng and 100 pg; 2 biological replicates for each amount. 9 mouse embryonic stem cell R1 cell line from ATCC SCRC 1011 samples: 1000 cells 100 cells and 10 cells; 3 biological replicates for each amount. 3 spike in RNA control add 0.001 fmol Gaussia luciferase/GLuc with m6A modification and 0.001 fmol Cypridina luciferase/CLuc without xxx modification into the 12 ng polyA selected RNAs related samples: 12 ng polyA selected RNA from mouse liver; 6 ng polyA selected RNA from mouse liver and 6 ng polyA selected RNA from wild type/WT mouse embryonic stem cells from Geula S. et al. Science 2015; and 6 ng polyA selected RNA from mouse liver and 6 ng polyA selected RNA from Mettl3 knock out/KO mouse embryonic stem cells from Geula S. et al. Science 2015. 12 single mouse oocyte and embryo samples: oocytes at germinal vesicle GV and metaphase II MII stages and embryos at zygote 2 cell 8 cell and…   pubmed:37349523   ZF zygote Input 10cell Rep2 GSM5599714   tissue:ZF zygote Input 10cell|developmental stage:Zygote|molecule subtype:rRNA free RNA ZF zygote Input 10cell Rep2 Quality of raw reads was assessed using FastQC v0.11.8 and sequencing adapters were trimmed using Cutadapt v1.8.1 if clear adapter signal was detected at the three primeend of the reads. There is no adapter removal for zebrafish zygote samples GSM5599713 GSM5599714 GSM5599715 GSM5599716 GSM5599717 GSM5599718 GSM5599719 GSM5599720 GSM5599721 GSM5599722 GSM5599723 GSM5599724 GSM5599725 mouse polyA selected RNA samples GSM5599726 GSM5599727 GSM5599728 GSM5599729 GSM5599730 GSM5599731 GSM5599732 GSM5599733 and mouse MII oocyte Input sample GSM5599734. Trimmed reads were aligned to the reference genome mm10 for mouse danRer11 for zebrafish using HISAT2 v2.1.0 with the parameter “ 5 8 no mixed no discordant”. Multiply aligned reads were discarded as indicated by HISAT2. PCR duplicates were removed using Samtools v1.9. The read pairs that overlapped with ribosomal RNAs were removed using BEDTools v2.28.0. Genome coverage bigWig files bin size = 10 bp normalized by RPKM were generated by deepTools v3.2.0 bamCoverage with the parameter “ bs 10 normalizeUsing RPKM”. Genome build: mm10 for mouse danRer11 for zebrafish Supplementary files format and content: The bigWig files are generated by deepTools v3.2.0 bamCoverage with the bin size=10bp and normalized by RPKM. ZF zygote Input 10cell For mouse GV oocyte collection 8 wpf C57BL6/N females were injected with 5 units U of pregnant mare serum gonadotropin PMSG. post 48 hours ovaries were dissected and oocytes were isolated by puncturing the follicles. The procedure was carried out in M2 medium Sigma supplemented with 0.2 mM of the cyclic nucleotide phosphodiesterase inhibitor 3 Isobutyl 1 methylxanthine IBMX Sigma to prevent the oocytes from further progress to germinal vesicle breakdown GVBD. The cumulus cells were gently removed by pipetting and the oocytes were briefly exposed to acidic Tyrode's solution Sigma to remove the zona pellucida followed by three washes in M2 medium. For mouse MII oocyte collection 4 wpf 5 wpf C57BL6/N females were injected with 5 U of PMSG followed by 5 U of human chorionic gonadotropin hCG 45 hours later. The oviducts were dissected 20 22 hours later and transferred to a clean dish containing M2 medium. The oviduct ampulla was identified under a stereomicroscope to isolate MII oocytes containing the cumulus mass. The oocytes were treated with 0.3 mg/ml hyaluronidase dissolved in M2 medium to remove the cumulus cells and then exposed to acidic Tyrode's solution for a few seconds to remove the zona pellucida followed by three washes in M2 medium. For mouse early embryo collection 8 wpf C57BL/6N females were superovulated by hormone injection 5 U PMSG followed by 5 U hCG 45 hours later and transferred to cages with 8 wpf C57BL/6N males for mating. At 27 28 hours zygote 39 43 hours 2 cell 68 70 hours 8 cell and 92 94 hours blastocyst post hCG administration the female mice were euthanized by cervical dislocation. Embryos were flushed from the reproductive tract into HEPES buffered CZB medium then transferred to acidic Tyrode's solution for a few seconds to remove the zona pellucida followed by three washes in M2 medium. For zebrafish bulk zygote samples total RNA was extracted from 100 zebrafish zygotes using TRIzol™ Reagent and eluated in 100 μl of RNase free water. Then 10 μl and 5 μl samples were taken for 10 zygote and 5 zygote samples respectively and volumes were adjusted to 12 μl with nuclease free water. For zebrafish single zygote samples single zygote was manually picked and distributed into 12 μl of 1X Lysis buffer Takara. For the polyA selected RNA samples of mouse liver and embryonic stem cell WT and Mettl3 KO total RNA was extracted using TRIzol™ Reagent Thermo Fisher Scientific and PolyA+ RNA was selected twice by Dynabeads™ mRNA Purification Kit Thermo Fisher Scientific. For mouse embryonic stem cell samples the cells were sorted into 12 μl 1X Lysis buffer using BD FACSMelody™ Cell Sorter BD Biosciences. For mouse oocyte and embryo samples post three washes in M2 medium oocytes and embryos were manually picked and put into 12 μl 1X Lysis buffer. For picoMeRIP seq experiment NEBNext® rRNA Depletion Kit NEB was used to delete DNA and rRNA; RNA samples were sheared by sonication incubated with the anti m6A antibody from Millipore ABE572 and precipitated using ethanol. SMART Seq® Stranded Kit Takara Cat. # 634442 was used to prepare RNA sequencing library. Libraries were quantified with KAPA Library Quantification Kits Roche and the size distribution was checked using TapeStation D1000 ScreenTape Agilent Technologies.   developmental stage:Zygote|molecule subtype:rRNA free RNA GSM5599714 GSM5599714: ZF zygote Input 10cell Rep2; Danio rerio; RIP Seq GSM5599714   1 For mouse liver total RNA was extracted using TRIzol™ Reagent Thermo Fisher Scientific and PolyA+ RNA was selected twice by Dynabeads™ mRNA Purification Kit Thermo Fisher Scientific. For zebrafish zygote mouse MII oocyte and mouse blastocyst samples the single cells were manually picked and sorted into 12 μl 1X lysis buffer Takara; NEBNext® rRNA Depletion Kit NEB was used to delete DNA and rRNA; RNA samples were sonicated for 2 × 30 seconds incubated with the anti m6A antibody from Millipore ABE572 and precipitated using ethanol. SMART Seq® Stranded Kit Takara Cat. # 634442 was used to prepare RNA sequencing library. Libraries were quantified with KAPA Library Quantification Kits Roche and the size distribution was checked using TapeStation D1000 ScreenTape Agilent Technologies. GEO Accession:GSM5599714 RIP-Seq TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina NovaSeq 6000   SRP339082   loader:fastq load.py ZF_zygote_Input_10cell_Rep2_R1.fastq.gz ZF_zygote_Input_10cell_Rep2_R2.fastq.gz fastq fastq 3842772582.0 37674241.0 GSM5599714 r1 0:51 1:51 A:843564828;C:1054412690;G:1130769812;T:813758353;N:266899 51 51     843564828 1054412690 1130769812 813758353 266899 SRX12382346 SRS10353008 SRA1301707 GEO Room 2035, Palmer Commons, Department of Computational Medicine and Bioinformatics, University of Michigan 2 0.91511 0.90868 0.32791 0.38048 0.89457 0.89441 0.81532 0.75274 51 51 B B biological fallback assumption illumina novaseq_era 3prime poly_a nebnext bulk bulk bulk   United States 2021-09-28 Zygote Embryo Oocyte Reproductive System
66206 66206 SRR16096221 SRX12382345 SRS10353007 SRP339082 PRJNA766854 RNA m6A methylome from zebrafish zygotes mouse liver polyA selected RNAs mouse embryonic stem cells spike in RNA controls and single mouse oocytes and embryos GSE184893 Other N6 methyladenosine m6A is a prevalent RNA modification that has key roles in distinct biological processes including meiosis and early embryo development. Despite advances in methodology for m6A mapping the applicability of current methods is limited by the need for large amounts of input material. We develop a sensitive picogram scale m6A RNA immunoprecipitation and sequencing picoMeRIP seq method also suitable for single cell scMeRIP seq. We validate the down scaling of the method by profiling m6A from picogram amounts of mouse liver polyA selected RNA mouse embryonic stem cells and zebrafish zygotes. We provide proof of principle of m6A profiling in single mouse oocytes and preimplantation embryos. Our work opens a new avenue for studying m6A in single cells and scarce cell types in a transcriptome wide manner. Overall design: Transcriptome wide m6A maps were generated from zebrafish and mouse samples as below. 11 zebrafish zygote samples: 3 biological replicates for 10 zygotes 3 biological replicates for 5 zygotes and 5 biological replicates for single zygote. 6 mouse liver polyA selected RNA samples: 10 ng 1 ng and 100 pg; 2 biological replicates for each amount. 9 mouse embryonic stem cell R1 cell line from ATCC SCRC 1011 samples: 1000 cells 100 cells and 10 cells; 3 biological replicates for each amount. 3 spike in RNA control add 0.001 fmol Gaussia luciferase/GLuc with m6A modification and 0.001 fmol Cypridina luciferase/CLuc without xxx modification into the 12 ng polyA selected RNAs related samples: 12 ng polyA selected RNA from mouse liver; 6 ng polyA selected RNA from mouse liver and 6 ng polyA selected RNA from wild type/WT mouse embryonic stem cells from Geula S. et al. Science 2015; and 6 ng polyA selected RNA from mouse liver and 6 ng polyA selected RNA from Mettl3 knock out/KO mouse embryonic stem cells from Geula S. et al. Science 2015. 12 single mouse oocyte and embryo samples: oocytes at germinal vesicle GV and metaphase II MII stages and embryos at zygote 2 cell 8 cell and…   pubmed:37349523   ZF zygote Input 10cell Rep1 GSM5599713   tissue:ZF zygote Input 10cell|developmental stage:Zygote|molecule subtype:rRNA free RNA ZF zygote Input 10cell Rep1 Quality of raw reads was assessed using FastQC v0.11.8 and sequencing adapters were trimmed using Cutadapt v1.8.1 if clear adapter signal was detected at the three primeend of the reads. There is no adapter removal for zebrafish zygote samples GSM5599713 GSM5599714 GSM5599715 GSM5599716 GSM5599717 GSM5599718 GSM5599719 GSM5599720 GSM5599721 GSM5599722 GSM5599723 GSM5599724 GSM5599725 mouse polyA selected RNA samples GSM5599726 GSM5599727 GSM5599728 GSM5599729 GSM5599730 GSM5599731 GSM5599732 GSM5599733 and mouse MII oocyte Input sample GSM5599734. Trimmed reads were aligned to the reference genome mm10 for mouse danRer11 for zebrafish using HISAT2 v2.1.0 with the parameter “ 5 8 no mixed no discordant”. Multiply aligned reads were discarded as indicated by HISAT2. PCR duplicates were removed using Samtools v1.9. The read pairs that overlapped with ribosomal RNAs were removed using BEDTools v2.28.0. Genome coverage bigWig files bin size = 10 bp normalized by RPKM were generated by deepTools v3.2.0 bamCoverage with the parameter “ bs 10 normalizeUsing RPKM”. Genome build: mm10 for mouse danRer11 for zebrafish Supplementary files format and content: The bigWig files are generated by deepTools v3.2.0 bamCoverage with the bin size=10bp and normalized by RPKM. ZF zygote Input 10cell For mouse GV oocyte collection 8 wpf C57BL6/N females were injected with 5 units U of pregnant mare serum gonadotropin PMSG. post 48 hours ovaries were dissected and oocytes were isolated by puncturing the follicles. The procedure was carried out in M2 medium Sigma supplemented with 0.2 mM of the cyclic nucleotide phosphodiesterase inhibitor 3 Isobutyl 1 methylxanthine IBMX Sigma to prevent the oocytes from further progress to germinal vesicle breakdown GVBD. The cumulus cells were gently removed by pipetting and the oocytes were briefly exposed to acidic Tyrode's solution Sigma to remove the zona pellucida followed by three washes in M2 medium. For mouse MII oocyte collection 4 wpf 5 wpf C57BL6/N females were injected with 5 U of PMSG followed by 5 U of human chorionic gonadotropin hCG 45 hours later. The oviducts were dissected 20 22 hours later and transferred to a clean dish containing M2 medium. The oviduct ampulla was identified under a stereomicroscope to isolate MII oocytes containing the cumulus mass. The oocytes were treated with 0.3 mg/ml hyaluronidase dissolved in M2 medium to remove the cumulus cells and then exposed to acidic Tyrode's solution for a few seconds to remove the zona pellucida followed by three washes in M2 medium. For mouse early embryo collection 8 wpf C57BL/6N females were superovulated by hormone injection 5 U PMSG followed by 5 U hCG 45 hours later and transferred to cages with 8 wpf C57BL/6N males for mating. At 27 28 hours zygote 39 43 hours 2 cell 68 70 hours 8 cell and 92 94 hours blastocyst post hCG administration the female mice were euthanized by cervical dislocation. Embryos were flushed from the reproductive tract into HEPES buffered CZB medium then transferred to acidic Tyrode's solution for a few seconds to remove the zona pellucida followed by three washes in M2 medium. For zebrafish bulk zygote samples total RNA was extracted from 100 zebrafish zygotes using TRIzol™ Reagent and eluated in 100 μl of RNase free water. Then 10 μl and 5 μl samples were taken for 10 zygote and 5 zygote samples respectively and volumes were adjusted to 12 μl with nuclease free water. For zebrafish single zygote samples single zygote was manually picked and distributed into 12 μl of 1X Lysis buffer Takara. For the polyA selected RNA samples of mouse liver and embryonic stem cell WT and Mettl3 KO total RNA was extracted using TRIzol™ Reagent Thermo Fisher Scientific and PolyA+ RNA was selected twice by Dynabeads™ mRNA Purification Kit Thermo Fisher Scientific. For mouse embryonic stem cell samples the cells were sorted into 12 μl 1X Lysis buffer using BD FACSMelody™ Cell Sorter BD Biosciences. For mouse oocyte and embryo samples post three washes in M2 medium oocytes and embryos were manually picked and put into 12 μl 1X Lysis buffer. For picoMeRIP seq experiment NEBNext® rRNA Depletion Kit NEB was used to delete DNA and rRNA; RNA samples were sheared by sonication incubated with the anti m6A antibody from Millipore ABE572 and precipitated using ethanol. SMART Seq® Stranded Kit Takara Cat. # 634442 was used to prepare RNA sequencing library. Libraries were quantified with KAPA Library Quantification Kits Roche and the size distribution was checked using TapeStation D1000 ScreenTape Agilent Technologies.   developmental stage:Zygote|molecule subtype:rRNA free RNA GSM5599713 GSM5599713: ZF zygote Input 10cell Rep1; Danio rerio; RIP Seq GSM5599713   1 For mouse liver total RNA was extracted using TRIzol™ Reagent Thermo Fisher Scientific and PolyA+ RNA was selected twice by Dynabeads™ mRNA Purification Kit Thermo Fisher Scientific. For zebrafish zygote mouse MII oocyte and mouse blastocyst samples the single cells were manually picked and sorted into 12 μl 1X lysis buffer Takara; NEBNext® rRNA Depletion Kit NEB was used to delete DNA and rRNA; RNA samples were sonicated for 2 × 30 seconds incubated with the anti m6A antibody from Millipore ABE572 and precipitated using ethanol. SMART Seq® Stranded Kit Takara Cat. # 634442 was used to prepare RNA sequencing library. Libraries were quantified with KAPA Library Quantification Kits Roche and the size distribution was checked using TapeStation D1000 ScreenTape Agilent Technologies. GEO Accession:GSM5599713 RIP-Seq TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina NovaSeq 6000   SRP339082   loader:fastq load.py ZF_zygote_Input_10cell_Rep1_R1.fastq.gz ZF_zygote_Input_10cell_Rep1_R2.fastq.gz fastq fastq 4029368016.0 39503608.0 GSM5599713 r1 0:51 1:51 A:893562237;C:1096986475;G:1180690796;T:857849967;N:278541 51 51     893562237 1096986475 1180690796 857849967 278541 SRX12382345 SRS10353007 SRA1301707 GEO Room 2035, Palmer Commons, Department of Computational Medicine and Bioinformatics, University of Michigan 2 0.91491 0.90807 0.32799 0.37836 0.89292 0.89282 0.81006 0.73354 51 51 B B biological fallback assumption illumina novaseq_era 3prime poly_a nebnext bulk bulk bulk   United States 2021-09-28 Zygote Embryo Oocyte Reproductive System

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