{"database": "metadata", "table": "run_metadata", "rows": [[49537, "SRR8937007", "SRX5717520", "SRS4655940", "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/\u00ce\u00bcl RNasin by rotating at 4\u00c2\u00b0C 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\u00c2\u00b0C for 1 h. 50 \u00ce\u00bcl 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 \u00ce\u00bcl Protein A Dynabeads for 4 h at 4\u00c2\u00b0C 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/\u00c2\u00b5l RNasin for eight times and once with 1ml ice cold 1\u00c3\u2014 PK buffer 100 mM Tris HCl pH 7.4  50 mM NaCl  10 mM EDTA   0.2% SDS  the beads was treated in 200 ul PK buffer containing 20 ul proteinase K Roche  0311582001 for 1 h at 55\u00c2\u00b0C. The solution was collected and subjected to RNA extraction with Acid Phenol: ChCl3 pH4.34.7 and ethanol precipitation. The Input RNA was extracted by using TRIzol reagent. Both the Input and IP RNA were treated by TURBO\u00e2\u201e\u00a2 DNase Invitrogen  AM2238. iCLIP seq: 1000 zebrafish embryos at 4 hpf were irradiated twice with 0.8 J/cm2 Stratalinker 2400  Stratagene  lysed and subjected to mild fragmentation. Crosslinked RNA protein complexes were immunoprecipated using polyclonal Ybx1 antibody Abmax and protein A dynabeads. 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. RNA RIP BisSeq: 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/\u00ce\u00bcl RNasin by rotating at 4\u00c2\u00b0C 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\u00c2\u00b0C for 1 h. 50 \u00ce\u00bcl 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 \u00ce\u00bcl Protein A Dynabeads for 4 h at 4\u00c2\u00b0C 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/\u00c2\u00b5l RNasin for eight times and once with 1ml ice cold 1\u00c3\u2014 PK buffer 100 mM Tris HCl pH 7.4  50 mM NaCl  10 mM EDTA   0.2% SDS  the beads was treated in 200 ul PK buffer containing 20 ul proteinase K Roche  0311582001 for 1 h at 55\u00c2\u00b0C. The solution was collected and subjected to RNA extraction with Acid Phenol: ChCl3 pH4.34.7 and ethanol precipitation. The purified mRNA was used for RNA BisSeq library construction. Around 200 ng mRNA premixed with the in vitro transcribed Dhfr mRNA at a ratio of 300:1 Dhfr mRNA serves as methylation conversion control was fragmented to 100 nt fragments for 1 min at 90\u00b0C in 10\u00d7 RNA Fragmentation Reagent Ambion  then stopped by 10\u00d7 RNA stop solution Ambion  and precipitated with 100% ethanol. The RNA pellet was resuspended in 100 \u00b5l bisulfite solution pH 5.1  which is a 100:1 mixture of 40% sodium bisulfite Sigma and 600 \u00b5M hydroquinone Sigma and subjected to heat incubation at 75\u00b0C for 4.5 h. The reaction mixture was desalted by passing through Nanosep with 3K Omega 500/pk columns PALL Corporation with centrifugation. post washed with nuclease free water and centrifuged for five times  the RNA was finally disolved in 75 ?l nuclease free water and then desulfonated by incubation with an equal volume of 1 M Tris HCl pH 9.0 at 75 \u00b0C for 1 h. post ethanol precipitation  the RNA was resuspended in 11 \u00b5l of RNase free water and subjected to library construction. Reverse transcription was carried out with superscript II Reverse Transcriptase Invitrogen and ACT random hexamers. The following procedures were performed with the KAPA Stranded mRNA Seq Kit KAPA according to the manufacturer?s instructions.Libraries were sequenced using HiSeq2500 Illumina in paired read mode  creating reads with a length of 125 bp.", "parent bioproject:PRJNA534030", null, null, "RIP BS 3hpf", "GSM3732427", null, "source name:Zebrafish embryo|strain:AB strain|age:3 hpf|tissue:whole embryo|rip antibody:polyclonal anti Ybx1 antibody", "RIP BS 3hpf", "library strategy: RNA RIP BisSeq Reads were aligned to the zv9 genome assembly using meRanTK v1.2.0 m5C sites were called by meRanCall v1.2.0 and annotated by applying BEDTools\u2019 intersectBed. MACS2 v2.1.1 were used for the peak calling and peaks were annotated by applying BEDTools\u2019 intersectBed. Genome build: zv9 Supplementary files format and content: Ybx1 binding m5C sites in one biological replicates.", "Zebrafish embryo", null, "Total RNA was isolated from zebrafish embryos harvested at different stages using TRIzol\u00ae Reagent Ambion. mRNA was extracted with using Dynabeads\u00ae mRNA Purification Kit Ambion and subjected to TURBO\u2122 DNase Invitrogen treatment at 37\u00b0C for 30 min and ethanol precipitation. Thus purified mRNA was used for RNA BisSeq library construction. Thus purified mRNA was used for RNA BisSeq library construction. Around 200 ng mRNA premixed with the in vitro transcribed Dhfr mRNA at a ratio of 300:1 Dhfr mRNA serves as methylation conversion control was fragmented to 100 nt fragments for 1 min at 90\u00b0C in 10\u00d7 RNA Fragmentation Reagent Ambion  then stopped by 10\u00d7 RNA stop solution Ambion  and precipitated with 100% ethanol. The RNA pellet was resuspended in 100 \u00b5l bisulfite solution pH 5.1  which is a 100:1 mixture of 40% sodium bisulfite Sigma and 600 \u00b5M hydroquinone Sigma and subjected to heat incubation at 75\u00b0C for 4.5 h. The reaction mixture was desalted by passing through Nanosep with 3K Omega 500/pk columns PALL Corporation with centrifugation. post washed with nuclease free water and centrifuged for five times  the RNA was finally disolved in 75 \u03bcl nuclease free water and then desulfonated by incubation with an equal volume of 1 M Tris HCl pH 9.0 at 75 \u00b0C for 1 h. post ethanol precipitation  the RNA was resuspended in 11 \u00b5l of RNase free water and subjected to library construction. Reverse transcription was carried out with superscript II Reverse Transcriptase Invitrogen and ACT random hexamers. The following procedures were performed with the KAPA Stranded mRNA Seq Kit KAPA according to the manufacturer\u2019s instructions.Libraries were sequenced using HiSeq2500 Illumina in paired read mode  creating reads with a length of 125 bp.", null, "strain:AB strain|age:3 hpf|tissue:whole embryo|rip antibody:polyclonal anti Ybx1 antibody", "GSM3732427", "GSM3732427: RIP BS 3hpf; Danio rerio; OTHER", "GSM3732427", null, "1", "Total RNA was isolated from zebrafish embryos harvested at different stages using TRIzol\u00ae Reagent Ambion. mRNA was extracted with using Dynabeads\u00ae mRNA Purification Kit Ambion and subjected to TURBO\u2122 DNase Invitrogen treatment at 37\u00b0C for 30 min and ethanol precipitation. Thus purified mRNA was used for RNA BisSeq library construction. Thus purified mRNA was used for RNA BisSeq library construction. Around 200 ng mRNA premixed with the in vitro transcribed Dhfr mRNA at a ratio of 300:1 Dhfr mRNA serves as methylation conversion control was fragmented to 100 nt fragments for 1 min at 90\u00b0C in 10\u00d7 RNA Fragmentation Reagent Ambion  then stopped by 10\u00d7 RNA stop solution Ambion  and precipitated with 100% ethanol. The RNA pellet was resuspended in 100 \u00b5l bisulfite solution pH 5.1  which is a 100:1 mixture of 40% sodium bisulfite Sigma and 600 \u00b5M hydroquinone Sigma and subjected to heat incubation at 75\u00b0C for 4.5 h. The reaction mixture was desalted by passing through Nanosep with 3K Omega 500/pk columns PALL Corporation with centrifugation. post washed with nuclease free water and centrifuged for five times  the RNA was finally disolved in 75 \u03bcl nuclease free water and then desulfonated by incubation with an equal volume of 1 M Tris HCl pH 9.0 at 75 \u00b0C for 1 h. post ethanol precipitation  the RNA was resuspended in 11 \u00b5l of RNase free water and subjected to library construction. Reverse transcription was carried out with superscript II Reverse Transcriptase Invitrogen and ACT random hexamers. The following procedures were performed with the KAPA Stranded mRNA Seq Kit KAPA according to the manufacturer's instructions.Libraries were sequenced using HiSeq2500 Illumina in paired read mode  creating reads with a length of 125 bp.", "GEO Accession:GSM3732427", "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "Illumina HiSeq 2500", null, "SRP162876", null, null, "RIP-BS_3hpf_R1.fastq.gz RIP-BS_3hpf_R2.fastq.gz", "fastq fastq", 46222984200.0, 154076614.0, "GSM3732427 r1", "0:150 1:150", "A:13069130635;C:10120441406;G:10858372086;T:12169793951;N:5246122", 150, 150, null, null, 13069130635, 10120441406, 10858372086, 12169793951, 5246122, "SRX5717520", "SRS4655940", "SRA786939", "GEO", "Beijing Institute of Genomics (BIG) of Chinese Academy of Sciences (CAS)", 2, 0.0063, 0.00488, 0.00135, 0.00112, 0.99736, 0.99801, 0.66703, 0.76167, 150, 150, "T", "T", "mates < 9% mapping rate", "illumina", "hiseq_era", "full_length", "random_priming", "smarter", "bulk", "clip", "iclip", null, "China", "2019-04-22", "Blastula", "Embryo", "Whole Organism", "All anatomical structures"]], "columns": ["rowid", "run.accession", "experiment.accession", "sample.accession", "study.accession", "bioproject", "study.title", "study.alias", "study.type", "study.abstract", "study.attributes", "study.PMIDs", "sample.description", "sample.title", "sample.alias", "sample.centername", "sample.attributes", "GEOsample.title", "GEOsample.dataprocessing", "GEOsample.source", "GEOsample.treatmentprotocol", "GEOsample.extractprotocol", "GEOsample.growthprotocol", "GEOsample.characteristics", "GEOsample.accession", "experiment.title", "experiment.alias", "experiment.library_name", "experiment.design_description", "experiment.library_construction_protocol", "experiment.attributes", "experiment.library_strategy", "experiment.library_source", "experiment.library_selection", "experiment.library_layout", "experiment.platform", "experiment.instrument_model", "experiment.spot_descriptor", "experiment.study_ref", "run.title", "run.attributes", "run.filename", "run.semantic_name", "run.total_bases", "run.total_spots", "run.alias", "run.read_lengths", "run.base_counts", "run.r1_length", "run.r2_length", "run.r3_length", "run.r4_length", "run.Acount", "run.Ccount", "run.Gcount", "run.Tcount", "run.Ncount", "run.experiment", "run.pool_member", "submission.accession", "submission.srasource", "submission.bioprojectsource", "seqdetective.n_mates", "seqdetective.mapping_rate.mate1", "seqdetective.mapping_rate.mate2", "seqdetective.nofeature_rate.mate1", "seqdetective.nofeature_rate.mate2", "seqdetective.sparsity.mate1", "seqdetective.sparsity.mate2", "seqdetective.pos_strand_rate.mate1", "seqdetective.pos_strand_rate.mate2", "seqdetective.readlen.mate1", "seqdetective.readlen.mate2", "seqdetective.judgement.mate1", "seqdetective.judgement.mate2", "seqdetective.judgement.reason", "platform_family", "instrument_generation", "read_bias", "selection_class", "prep_kit", "sc_or_bulk", "tech_class", "technology", "tech_variant", "submission.bioprojectsource.country", "earliest_date", "devstage_curation", "devstage_curation_coarse", "tissue_curation", "tissue_curation_coarse"], "primary_keys": ["rowid"], "primary_key_values": ["49537"], "units": {}, "query_ms": 11.159739000504487}