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
41527,SRR5441898,SRX2731753,SRS2119651,SRP092907,PRJNA352850,Transcriptomics analysis of gene expressions  m6A enrichment levels and ythdf2 binding targets in control and mettl3 or ythdf2 morphants zebrafish embryos,GSE89655,Other,RNA was isolated from control and mettl3 or ythdf2 morphants zebrafish cells using the TRIzol Invitrogen reagent by following the company manual. For all samples the RNA integrity was checked using an Agilent Bioanalyzer 2100. All samples showed a RIN RNA integrity number of higher than 9. Approximately 2.5 µg of total RNA was then used for library preparation using a TruSeq™ RNA Sample Prep Kit v2 Illumina  San Diego  CA  USA according to the manufacturer’s protocol.The libraries were sequenced using HiSeq2500 or HiSeq 3000 Illumina in paired read mode  creating reads with a length of 101 or 151 bp. Sequencing chemistry v2 Illumina was used. Overall design: Examination of gene expressive levels  m6A enrichment levels  m6A single uncleotide sites and ythdf2 binding targets in normal and mettl3 or ythdf2 morphants zebrafish cells,,pubmed:28869969,,control zebrafish embryos m6A miCLIP rep2,GSM2572320,,source name:zebrafish embryos|cell type:normal zebrafish embryos cells|age:28hpf|tissue:trunk|enrichment antibody vendor:Abcam,control zebrafish embryos m6A miCLIP rep2,library strategy: miCLIP seq Reads were trimmed for adaptor sequence using fastx clipper from FASTX Toolkit fastx clipper  a AGATCGGAAGAGCACACG  n  Q 33. Low quality bases were filtered by fastq filter.pl  a custom perl script from CLIP Tool Kit CTK and reads shorter than 24 nt would be discard. The forward reads were demultiplexed based on 5’ barcodes for individual replicates by fastq2collapse.pl to remove PCR amplified reads. The reverse reads were reversed complemented and processed like their forward mates. And then paired end reads were mixed for downstream analysis. Random barcodes of remained reads were stripped by stripBarcode.pl. Then the barcode sequence was moved to header line for each read. Remained reads were mapped to the zebrafish genomes version zv9 with BWA v0.7.10. The CIMS pipeline https://zhanglab.c2b2.columbia.edu/index.php/CTK Documentation was used to collapse PCR duplicates based on their mapped coordinates and to determine the unique tag coverage k and the number of mutation m for each nucleotide. The filter for the dataset was k >= 15 and m/k <= 50% and only mutation positions within RRACH motif were identified as m6A for downstream analysis to exclude the potential m6Am modification. Genome build: zv9 Supplementary files format and content: Single nucleotide resolution m6A sites identified by CIMS software.,zebrafish embryos,,For miCLIP seq  mRNA was isolate from the trunck region of zebrafish embryos at 28hpf and the RNA integrity was checked using an Agilent Bioanalyzer 2100. 10 ug of mRNA was first fragmented and immunoprecipitated with m6A antibody and thus obtained m6A containing RNA was subjected to cDNA library construction according to previously reported method [Linder et al. 2015; PMID: 26121403]. The libraries were sequenced using HiSeq3000 Illumina in paired read mode  creating reads with a length of 101 bp.,,cell type:normal zebrafish embryos cells|age:28hpf|tissue:trunk|enrichment antibody vendor:Abcam,GSM2572320,GSM2572320: control zebrafish embryos m6A miCLIP rep2; Danio rerio; OTHER,GSM2572320,,1,For miCLIP seq  mRNA was isolate from the trunck region of zebrafish embryos at 28hpf and the RNA integrity was checked using an Agilent Bioanalyzer 2100. 10 ug of mRNA was first fragmented and immunoprecipitated with m6A antibody and thus obtained m6A containing RNA was subjected to cDNA library construction according to previously reported method [Linder et al. 2015; PMID: 26121403]. The libraries were sequenced using HiSeq2000 or HiSeq3000 Illumina in single read or paired read mode  creating reads with a length of 101 bp,GEO Accession:GSM2572320,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,Illumina HiSeq 3000,,SRP092907,,,miCLIP_Abcam_rep2_1.fastq.gz miCLIP_Abcam_rep2_2.fastq.gz,fastq fastq,5381144865.0,26771865.0,GSM2572320 r1,0:101 1:100,A:1590037266;C:1121098903;G:1187084038;T:1482144584;N:780074,101,100,,,1590037266,1121098903,1187084038,1482144584,780074,SRX2731753,SRS2119651,SRA491654,GEO,Beijing Institute of Genomics (BIG) of Chinese Academy of Sciences (CAS),2,0.55442,0.60543,0.10037,0.11559,0.75432,0.747,0.55512,0.44818,101,100,B,B,biological fallback assumption,illumina,hiseq_era,unknown,other,trueseq,bulk,clip,iclip,,China,2017-04-10,Pharyngula,Embryo,Trunk,Surface Structure
41528,SRR5441897,SRX2731752,SRS2119650,SRP092907,PRJNA352850,Transcriptomics analysis of gene expressions  m6A enrichment levels and ythdf2 binding targets in control and mettl3 or ythdf2 morphants zebrafish embryos,GSE89655,Other,RNA was isolated from control and mettl3 or ythdf2 morphants zebrafish cells using the TRIzol Invitrogen reagent by following the company manual. For all samples the RNA integrity was checked using an Agilent Bioanalyzer 2100. All samples showed a RIN RNA integrity number of higher than 9. Approximately 2.5 µg of total RNA was then used for library preparation using a TruSeq™ RNA Sample Prep Kit v2 Illumina  San Diego  CA  USA according to the manufacturer’s protocol.The libraries were sequenced using HiSeq2500 or HiSeq 3000 Illumina in paired read mode  creating reads with a length of 101 or 151 bp. Sequencing chemistry v2 Illumina was used. Overall design: Examination of gene expressive levels  m6A enrichment levels  m6A single uncleotide sites and ythdf2 binding targets in normal and mettl3 or ythdf2 morphants zebrafish cells,,pubmed:28869969,,control zebrafish embryos m6A miCLIP rep1,GSM2572319,,source name:zebrafish embryos|cell type:normal zebrafish embryos cells|age:28hpf|tissue:trunk|enrichment antibody vendor:Abcam,control zebrafish embryos m6A miCLIP rep1,library strategy: miCLIP seq Reads were trimmed for adaptor sequence using fastx clipper from FASTX Toolkit fastx clipper  a AGATCGGAAGAGCACACG  n  Q 33. Low quality bases were filtered by fastq filter.pl  a custom perl script from CLIP Tool Kit CTK and reads shorter than 24 nt would be discard. The forward reads were demultiplexed based on 5’ barcodes for individual replicates by fastq2collapse.pl to remove PCR amplified reads. The reverse reads were reversed complemented and processed like their forward mates. And then paired end reads were mixed for downstream analysis. Random barcodes of remained reads were stripped by stripBarcode.pl. Then the barcode sequence was moved to header line for each read. Remained reads were mapped to the zebrafish genomes version zv9 with BWA v0.7.10. The CIMS pipeline https://zhanglab.c2b2.columbia.edu/index.php/CTK Documentation was used to collapse PCR duplicates based on their mapped coordinates and to determine the unique tag coverage k and the number of mutation m for each nucleotide. The filter for the dataset was k >= 15 and m/k <= 50% and only mutation positions within RRACH motif were identified as m6A for downstream analysis to exclude the potential m6Am modification. Genome build: zv9 Supplementary files format and content: Single nucleotide resolution m6A sites identified by CIMS software.,zebrafish embryos,,For miCLIP seq  mRNA was isolate from the trunck region of zebrafish embryos at 28hpf and the RNA integrity was checked using an Agilent Bioanalyzer 2100. 10 ug of mRNA was first fragmented and immunoprecipitated with m6A antibody and thus obtained m6A containing RNA was subjected to cDNA library construction according to previously reported method [Linder et al. 2015; PMID: 26121403]. The libraries were sequenced using HiSeq3000 Illumina in paired read mode  creating reads with a length of 101 bp.,,cell type:normal zebrafish embryos cells|age:28hpf|tissue:trunk|enrichment antibody vendor:Abcam,GSM2572319,GSM2572319: control zebrafish embryos m6A miCLIP rep1; Danio rerio; OTHER,GSM2572319,,1,For miCLIP seq  mRNA was isolate from the trunck region of zebrafish embryos at 28hpf and the RNA integrity was checked using an Agilent Bioanalyzer 2100. 10 ug of mRNA was first fragmented and immunoprecipitated with m6A antibody and thus obtained m6A containing RNA was subjected to cDNA library construction according to previously reported method [Linder et al. 2015; PMID: 26121403]. The libraries were sequenced using HiSeq2000 or HiSeq3000 Illumina in single read or paired read mode  creating reads with a length of 101 bp,GEO Accession:GSM2572319,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,Illumina HiSeq 3000,,SRP092907,,,miCLIP_Abcam_rep1_1.fastq.gz miCLIP_Abcam_rep1_2.fastq.gz,fastq fastq,6036174318.0,30030718.0,GSM2572319 r1,0:101 1:100,A:1726466495;C:1297674104;G:1364186585;T:1647084426;N:762708,101,100,,,1726466495,1297674104,1364186585,1647084426,762708,SRX2731752,SRS2119650,SRA491654,GEO,Beijing Institute of Genomics (BIG) of Chinese Academy of Sciences (CAS),2,0.5985,0.66835,0.09404,0.1086,0.74491,0.73596,0.54343,0.45047,101,100,B,B,biological fallback assumption,illumina,hiseq_era,unknown,other,trueseq,bulk,clip,iclip,,China,2017-04-10,Pharyngula,Embryo,Trunk,Surface Structure
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/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 in 200 ul PK buffer containing 20 ul proteinase K Roche  0311582001 for 1 h at 55°C. 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 TURBOâ„¢ 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/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 in 200 ul PK buffer containing 20 ul proteinase K Roche  0311582001 for 1 h at 55°C. 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°C in 10× RNA Fragmentation Reagent Ambion  then stopped by 10× RNA stop solution Ambion  and precipitated with 100% ethanol. The RNA pellet was resuspended in 100 µl bisulfite solution pH 5.1  which is a 100:1 mixture of 40% sodium bisulfite Sigma and 600 µM hydroquinone Sigma and subjected to heat incubation at 75°C 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 °C for 1 h. post ethanol precipitation  the RNA was resuspended in 11 µl 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,,,RIP BS 3hpf,GSM3732427,,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’ intersectBed. 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 m5C sites in one biological replicates.,Zebrafish embryo,,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. 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°C in 10× RNA Fragmentation Reagent Ambion  then stopped by 10× RNA stop solution Ambion  and precipitated with 100% ethanol. The RNA pellet was resuspended in 100 µl bisulfite solution pH 5.1  which is a 100:1 mixture of 40% sodium bisulfite Sigma and 600 µM hydroquinone Sigma and subjected to heat incubation at 75°C 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 μl nuclease free water and then desulfonated by incubation with an equal volume of 1 M Tris HCl pH 9.0 at 75 °C for 1 h. post ethanol precipitation  the RNA was resuspended in 11 µl 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.,,strain:AB strain|age:3 hpf|tissue:whole embryo|rip antibody:polyclonal anti Ybx1 antibody,GSM3732427,GSM3732427: RIP BS 3hpf; Danio rerio; OTHER,GSM3732427,,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. 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°C in 10× RNA Fragmentation Reagent Ambion  then stopped by 10× RNA stop solution Ambion  and precipitated with 100% ethanol. The RNA pellet was resuspended in 100 µl bisulfite solution pH 5.1  which is a 100:1 mixture of 40% sodium bisulfite Sigma and 600 µM hydroquinone Sigma and subjected to heat incubation at 75°C 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 μl nuclease free water and then desulfonated by incubation with an equal volume of 1 M Tris HCl pH 9.0 at 75 °C for 1 h. post ethanol precipitation  the RNA was resuspended in 11 µl 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,,SRP162876,,,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,,,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,,China,2019-04-22,Blastula,Embryo,Whole Organism,All anatomical structures
49538,SRR8937006,SRX5717519,SRS4655939,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 in 200 ul PK buffer containing 20 ul proteinase K Roche  0311582001 for 1 h at 55°C. 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 TURBOâ„¢ 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/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 in 200 ul PK buffer containing 20 ul proteinase K Roche  0311582001 for 1 h at 55°C. 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°C in 10× RNA Fragmentation Reagent Ambion  then stopped by 10× RNA stop solution Ambion  and precipitated with 100% ethanol. The RNA pellet was resuspended in 100 µl bisulfite solution pH 5.1  which is a 100:1 mixture of 40% sodium bisulfite Sigma and 600 µM hydroquinone Sigma and subjected to heat incubation at 75°C 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 °C for 1 h. post ethanol precipitation  the RNA was resuspended in 11 µl 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,,,RIP BS 0hpf,GSM3732426,,source name:Zebrafish embryo|strain:AB strain|age:0 hpf|tissue:whole embryo|rip antibody:polyclonal anti Ybx1 antibody,RIP BS 0hpf,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’ intersectBed. 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 m5C sites in one biological replicates.,Zebrafish embryo,,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. 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°C in 10× RNA Fragmentation Reagent Ambion  then stopped by 10× RNA stop solution Ambion  and precipitated with 100% ethanol. The RNA pellet was resuspended in 100 µl bisulfite solution pH 5.1  which is a 100:1 mixture of 40% sodium bisulfite Sigma and 600 µM hydroquinone Sigma and subjected to heat incubation at 75°C 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 μl nuclease free water and then desulfonated by incubation with an equal volume of 1 M Tris HCl pH 9.0 at 75 °C for 1 h. post ethanol precipitation  the RNA was resuspended in 11 µl 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.,,strain:AB strain|age:0 hpf|tissue:whole embryo|rip antibody:polyclonal anti Ybx1 antibody,GSM3732426,GSM3732426: RIP BS 0hpf; Danio rerio; OTHER,GSM3732426,,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. 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°C in 10× RNA Fragmentation Reagent Ambion  then stopped by 10× RNA stop solution Ambion  and precipitated with 100% ethanol. The RNA pellet was resuspended in 100 µl bisulfite solution pH 5.1  which is a 100:1 mixture of 40% sodium bisulfite Sigma and 600 µM hydroquinone Sigma and subjected to heat incubation at 75°C 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 μl nuclease free water and then desulfonated by incubation with an equal volume of 1 M Tris HCl pH 9.0 at 75 °C for 1 h. post ethanol precipitation  the RNA was resuspended in 11 µl 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:GSM3732426,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,Illumina HiSeq 2500,,SRP162876,,,RIP-BS_0hpf_R1.fastq.gz RIP-BS_0hpf_R2.fastq.gz,fastq fastq,41224496700.0,137414989.0,GSM3732426 r1,0:150 1:150,A:11515809359;C:9072060634;G:9856717145;T:10775122637;N:4786925,150,150,,,11515809359,9072060634,9856717145,10775122637,4786925,SRX5717519,SRS4655939,SRA786939,GEO,Beijing Institute of Genomics (BIG) of Chinese Academy of Sciences (CAS),2,0.00575,0.00421,0.00122,0.00083,0.99738,0.99801,0.70652,0.78053,150,150,T,T,mates < 9% mapping rate,illumina,hiseq_era,full_length,random_priming,smarter,bulk,clip,iclip,,China,2019-04-22,Zygote,Embryo,Whole Organism,All anatomical structures
49539,SRR8937005,SRX5717518,SRS4655938,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 in 200 ul PK buffer containing 20 ul proteinase K Roche  0311582001 for 1 h at 55°C. 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 TURBOâ„¢ 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/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 in 200 ul PK buffer containing 20 ul proteinase K Roche  0311582001 for 1 h at 55°C. 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°C in 10× RNA Fragmentation Reagent Ambion  then stopped by 10× RNA stop solution Ambion  and precipitated with 100% ethanol. The RNA pellet was resuspended in 100 µl bisulfite solution pH 5.1  which is a 100:1 mixture of 40% sodium bisulfite Sigma and 600 µM hydroquinone Sigma and subjected to heat incubation at 75°C 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 °C for 1 h. post ethanol precipitation  the RNA was resuspended in 11 µl 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,,,RIP INPUT 2hpf rep2,GSM3732425,,source name:RIP INPUT 2hpf|strain:AB strain|age:2 hpf|tissue:whole embryo,RIP INPUT 2hpf 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 2hpf,,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:2 hpf|tissue:whole embryo,GSM3732425,GSM3732425: RIP INPUT 2hpf rep2; Danio rerio; RIP Seq,GSM3732425,,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:GSM3732425,RIP-Seq,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,Illumina HiSeq 2500,,SRP162876,,,RIP-INPUT_2hpf_rep2_R1.fastq.gz RIP-INPUT_2hpf_rep2_R2.fastq.gz,fastq fastq,16388119500.0,54627065.0,GSM3732425 r1,0:150 1:150,A:3232127065;C:4976385987;G:5052844082;T:3122288663;N:4473703,150,150,,,3232127065,4976385987,5052844082,3122288663,4473703,SRX5717518,SRS4655938,SRA786939,GEO,Beijing Institute of Genomics (BIG) of Chinese Academy of Sciences (CAS),2,0.98209,0.9812,0.33332,0.33565,0.93811,0.94186,0.95153,0.95984,150,150,B,B,biological fallback assumption,illumina,hiseq_era,full_length,random_priming,smarter,bulk,clip,iclip,,China,2019-04-22,Cleavage,Embryo,Whole Organism,All anatomical structures
49540,SRR8937004,SRX5717517,SRS4655937,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 in 200 ul PK buffer containing 20 ul proteinase K Roche  0311582001 for 1 h at 55°C. 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 TURBOâ„¢ 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/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 in 200 ul PK buffer containing 20 ul proteinase K Roche  0311582001 for 1 h at 55°C. 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°C in 10× RNA Fragmentation Reagent Ambion  then stopped by 10× RNA stop solution Ambion  and precipitated with 100% ethanol. The RNA pellet was resuspended in 100 µl bisulfite solution pH 5.1  which is a 100:1 mixture of 40% sodium bisulfite Sigma and 600 µM hydroquinone Sigma and subjected to heat incubation at 75°C 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 °C for 1 h. post ethanol precipitation  the RNA was resuspended in 11 µl 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,,,RIP INPUT 2hpf rep1,GSM3732424,,source name:RIP INPUT 2hpf|strain:AB strain|age:2 hpf|tissue:whole embryo,RIP INPUT 2hpf 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 2hpf,,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:2 hpf|tissue:whole embryo,GSM3732424,GSM3732424: RIP INPUT 2hpf rep1; Danio rerio; RIP Seq,GSM3732424,,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:GSM3732424,RIP-Seq,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,Illumina HiSeq 2500,,SRP162876,,,RIP-INPUT_2hpf_rep1_R1.fastq.gz RIP-INPUT_2hpf_rep1_R2.fastq.gz,fastq fastq,18857336400.0,62857788.0,GSM3732424 r1,0:150 1:150,A:3116761123;C:6239715125;G:6450542871;T:3049605751;N:711530,150,150,,,3116761123,6239715125,6450542871,3049605751,711530,SRX5717517,SRS4655937,SRA786939,GEO,Beijing Institute of Genomics (BIG) of Chinese Academy of Sciences (CAS),2,0.97874,0.96891,0.03494,0.03355,0.92693,0.93456,0.9502,0.94986,150,150,B,B,biological fallback assumption,illumina,hiseq_era,full_length,random_priming,smarter,bulk,clip,iclip,,China,2019-04-22,Cleavage,Embryo,Whole Organism,All anatomical structures
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 in 200 ul PK buffer containing 20 ul proteinase K Roche  0311582001 for 1 h at 55°C. 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 TURBOâ„¢ 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/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 in 200 ul PK buffer containing 20 ul proteinase K Roche  0311582001 for 1 h at 55°C. 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°C in 10× RNA Fragmentation Reagent Ambion  then stopped by 10× RNA stop solution Ambion  and precipitated with 100% ethanol. The RNA pellet was resuspended in 100 µl bisulfite solution pH 5.1  which is a 100:1 mixture of 40% sodium bisulfite Sigma and 600 µM hydroquinone Sigma and subjected to heat incubation at 75°C 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 °C for 1 h. post ethanol precipitation  the RNA was resuspended in 11 µl 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,,,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,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 in 200 ul PK buffer containing 20 ul proteinase K Roche  0311582001 for 1 h at 55°C. 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 TURBOâ„¢ 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/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 in 200 ul PK buffer containing 20 ul proteinase K Roche  0311582001 for 1 h at 55°C. 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°C in 10× RNA Fragmentation Reagent Ambion  then stopped by 10× RNA stop solution Ambion  and precipitated with 100% ethanol. The RNA pellet was resuspended in 100 µl bisulfite solution pH 5.1  which is a 100:1 mixture of 40% sodium bisulfite Sigma and 600 µM hydroquinone Sigma and subjected to heat incubation at 75°C 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 °C for 1 h. post ethanol precipitation  the RNA was resuspended in 11 µl 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,,,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
49543,SRR8937001,SRX5717514,SRS4655934,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 in 200 ul PK buffer containing 20 ul proteinase K Roche  0311582001 for 1 h at 55°C. 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 TURBOâ„¢ 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/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 in 200 ul PK buffer containing 20 ul proteinase K Roche  0311582001 for 1 h at 55°C. 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°C in 10× RNA Fragmentation Reagent Ambion  then stopped by 10× RNA stop solution Ambion  and precipitated with 100% ethanol. The RNA pellet was resuspended in 100 µl bisulfite solution pH 5.1  which is a 100:1 mixture of 40% sodium bisulfite Sigma and 600 µM hydroquinone Sigma and subjected to heat incubation at 75°C 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 °C for 1 h. post ethanol precipitation  the RNA was resuspended in 11 µl 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,,,RIP 2hpf rep2,GSM3732421,,source name:RIP 2hpf|strain:AB strain|age:2 hpf|tissue:whole embryo|rip antibody:rabbit polyclonal anti Ybx1 antibody,RIP 2hpf 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 2hpf,,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:2 hpf|tissue:whole embryo|rip antibody:rabbit polyclonal anti Ybx1 antibody,GSM3732421,GSM3732421: RIP 2hpf rep2; Danio rerio; RIP Seq,GSM3732421,,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:GSM3732421,RIP-Seq,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,Illumina HiSeq 2500,,SRP162876,,,RIP_2hpf_rep2_R1.fastq.gz RIP_2hpf_rep2_R2.fastq.gz,fastq fastq,17557024800.0,58523416.0,GSM3732421 r1,0:150 1:150,A:4454737772;C:4407385601;G:4364085079;T:4328962997;N:1853351,150,150,,,4454737772,4407385601,4364085079,4328962997,1853351,SRX5717514,SRS4655934,SRA786939,GEO,Beijing Institute of Genomics (BIG) of Chinese Academy of Sciences (CAS),2,0.96216,0.96226,0.08174,0.08009,0.76907,0.77693,0.68824,0.65939,150,150,B,B,biological fallback assumption,illumina,hiseq_era,full_length,random_priming,smarter,bulk,clip,iclip,,China,2019-04-22,Cleavage,Embryo,Whole Organism,All anatomical structures
49544,SRR8937000,SRX5717513,SRS4655933,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 in 200 ul PK buffer containing 20 ul proteinase K Roche  0311582001 for 1 h at 55°C. 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 TURBOâ„¢ 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/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 in 200 ul PK buffer containing 20 ul proteinase K Roche  0311582001 for 1 h at 55°C. 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°C in 10× RNA Fragmentation Reagent Ambion  then stopped by 10× RNA stop solution Ambion  and precipitated with 100% ethanol. The RNA pellet was resuspended in 100 µl bisulfite solution pH 5.1  which is a 100:1 mixture of 40% sodium bisulfite Sigma and 600 µM hydroquinone Sigma and subjected to heat incubation at 75°C 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 °C for 1 h. post ethanol precipitation  the RNA was resuspended in 11 µl 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,,,RIP 2hpf rep1,GSM3732420,,source name:RIP 2hpf|strain:AB strain|age:2 hpf|tissue:whole embryo|rip antibody:rabbit polyclonal anti Ybx1 antibody,RIP 2hpf 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 2hpf,,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:2 hpf|tissue:whole embryo|rip antibody:rabbit polyclonal anti Ybx1 antibody,GSM3732420,GSM3732420: RIP 2hpf rep1; Danio rerio; RIP Seq,GSM3732420,,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:GSM3732420,RIP-Seq,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,Illumina HiSeq 2500,,SRP162876,,,RIP_2hpf_rep1_R1.fastq.gz RIP_2hpf_rep1_R2.fastq.gz,fastq fastq,5656083600.0,18853612.0,GSM3732420 r1,0:150 1:150,A:1244757394;C:1581273853;G:1666307178;T:1162885398;N:859777,150,150,,,1244757394,1581273853,1666307178,1162885398,859777,SRX5717513,SRS4655933,SRA786939,GEO,Beijing Institute of Genomics (BIG) of Chinese Academy of Sciences (CAS),2,0.80705,0.80575,0.2454,0.24923,0.85478,0.85644,0.83319,0.80065,150,150,B,B,biological fallback assumption,illumina,hiseq_era,full_length,random_priming,smarter,bulk,clip,iclip,,China,2019-04-22,Cleavage,Embryo,Whole Organism,All anatomical structures
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 in 200 ul PK buffer containing 20 ul proteinase K Roche  0311582001 for 1 h at 55°C. 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 TURBOâ„¢ 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/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 in 200 ul PK buffer containing 20 ul proteinase K Roche  0311582001 for 1 h at 55°C. 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°C in 10× RNA Fragmentation Reagent Ambion  then stopped by 10× RNA stop solution Ambion  and precipitated with 100% ethanol. The RNA pellet was resuspended in 100 µl bisulfite solution pH 5.1  which is a 100:1 mixture of 40% sodium bisulfite Sigma and 600 µM hydroquinone Sigma and subjected to heat incubation at 75°C 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 °C for 1 h. post ethanol precipitation  the RNA was resuspended in 11 µl 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,,,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,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 in 200 ul PK buffer containing 20 ul proteinase K Roche  0311582001 for 1 h at 55°C. 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 TURBOâ„¢ 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/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 in 200 ul PK buffer containing 20 ul proteinase K Roche  0311582001 for 1 h at 55°C. 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°C in 10× RNA Fragmentation Reagent Ambion  then stopped by 10× RNA stop solution Ambion  and precipitated with 100% ethanol. The RNA pellet was resuspended in 100 µl bisulfite solution pH 5.1  which is a 100:1 mixture of 40% sodium bisulfite Sigma and 600 µM hydroquinone Sigma and subjected to heat incubation at 75°C 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 °C for 1 h. post ethanol precipitation  the RNA was resuspended in 11 µl 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,,,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
49547,SRR7942638,SRX4776903,SRS3857464,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 in 200 ul PK buffer containing 20 ul proteinase K Roche  0311582001 for 1 h at 55°C. 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 TURBOâ„¢ 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/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 in 200 ul PK buffer containing 20 ul proteinase K Roche  0311582001 for 1 h at 55°C. 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°C in 10× RNA Fragmentation Reagent Ambion  then stopped by 10× RNA stop solution Ambion  and precipitated with 100% ethanol. The RNA pellet was resuspended in 100 µl bisulfite solution pH 5.1  which is a 100:1 mixture of 40% sodium bisulfite Sigma and 600 µM hydroquinone Sigma and subjected to heat incubation at 75°C 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 °C for 1 h. post ethanol precipitation  the RNA was resuspended in 11 µl 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,,,iCLIP 4hpf rep2,GSM3406904,,source name:Zebrafish embryo|strain:AB strain|cell type:Zebrafish embryo|age:4 hpf|tissue:whole embryo,iCLIP 4hpf rep2,RIP seq and iCLIP seq reads were aligned to the zv9 genome assembly using TopHat v2.1.1 and BWA v0.7.10  respectively. RIP seq: MACS2 v2.1.1 were used for the peak calling and peaks were annotated by applying BEDTools’ intersectBed. iCLIP: To identify the Ybx1 targets’ locus  the mode of truncation calling was performed. For each truncation position  the coverages of unique tag k and truncations m were determined by CIMS.pl program CLIP Tool Kit  CTK. The sites were annotated by applying BEDTools’ intersectBed. Genome build: zv9 Supplementary files format and content: Ybx1 binding sites in two biological replicates.,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 in 200 ul PK buffer containing 20 ul proteinase K Roche  0311582001 for 1 h at 55°C. 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™ 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.,,strain:AB strain|cell type:Zebrafish embryo|age:4 hpf|tissue:whole embryo,GSM3406904,GSM3406904: iCLIP 4hpf rep2; Danio rerio; OTHER,GSM3406904,,1,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 in 200 ul PK buffer containing 20 ul proteinase K Roche  0311582001 for 1 h at 55°C. 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™ 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.,GEO Accession:GSM3406904,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,Illumina HiSeq 2500,,SRP162876,,,iCLIP_4hpf_rep2_R1.fastq.gz iCLIP_4hpf_rep2_R2.fastq.gz,fastq fastq,17432004900.0,58106683.0,GSM3406904 r1,0:150 1:150,A:5027776225;C:3413769313;G:3531157280;T:5459126811;N:175271,150,150,,,5027776225,3413769313,3531157280,5459126811,175271,SRX4776903,SRS3857464,SRA786939,GEO,Beijing Institute of Genomics (BIG) of Chinese Academy of Sciences (CAS),2,0.62059,0.61563,0.02838,0.02072,0.95298,0.9628,0.94729,0.94715,150,150,B,B,biological fallback assumption,illumina,hiseq_era,full_length,random_priming,smarter,bulk,clip,iclip,,China,2018-09-28,Blastula,Embryo,Whole Organism,All anatomical structures
49548,SRR7942637,SRX4776902,SRS3857447,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 in 200 ul PK buffer containing 20 ul proteinase K Roche  0311582001 for 1 h at 55°C. 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 TURBOâ„¢ 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/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 in 200 ul PK buffer containing 20 ul proteinase K Roche  0311582001 for 1 h at 55°C. 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°C in 10× RNA Fragmentation Reagent Ambion  then stopped by 10× RNA stop solution Ambion  and precipitated with 100% ethanol. The RNA pellet was resuspended in 100 µl bisulfite solution pH 5.1  which is a 100:1 mixture of 40% sodium bisulfite Sigma and 600 µM hydroquinone Sigma and subjected to heat incubation at 75°C 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 °C for 1 h. post ethanol precipitation  the RNA was resuspended in 11 µl 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,,,iCLIP 4hpf rep1,GSM3406903,,source name:Zebrafish embryo|strain:AB strain|cell type:Zebrafish embryo|age:4 hpf|tissue:whole embryo,iCLIP 4hpf rep1,RIP seq and iCLIP seq reads were aligned to the zv9 genome assembly using TopHat v2.1.1 and BWA v0.7.10  respectively. RIP seq: MACS2 v2.1.1 were used for the peak calling and peaks were annotated by applying BEDTools’ intersectBed. iCLIP: To identify the Ybx1 targets’ locus  the mode of truncation calling was performed. For each truncation position  the coverages of unique tag k and truncations m were determined by CIMS.pl program CLIP Tool Kit  CTK. The sites were annotated by applying BEDTools’ intersectBed. Genome build: zv9 Supplementary files format and content: Ybx1 binding sites in two biological replicates.,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 in 200 ul PK buffer containing 20 ul proteinase K Roche  0311582001 for 1 h at 55°C. 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™ 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.,,strain:AB strain|cell type:Zebrafish embryo|age:4 hpf|tissue:whole embryo,GSM3406903,GSM3406903: iCLIP 4hpf rep1; Danio rerio; OTHER,GSM3406903,,1,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 in 200 ul PK buffer containing 20 ul proteinase K Roche  0311582001 for 1 h at 55°C. 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™ 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.,GEO Accession:GSM3406903,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,Illumina HiSeq 2500,,SRP162876,,,iCLIP_4hpf_rep1_R1.fastq.gz iCLIP_4hpf_rep1_R2.fastq.gz,fastq fastq,18547407600.0,61824692.0,GSM3406903 r1,0:150 1:150,A:5264017245;C:3699523947;G:3688768985;T:5894912242;N:185181,150,150,,,5264017245,3699523947,3688768985,5894912242,185181,SRX4776902,SRS3857447,SRA786939,GEO,Beijing Institute of Genomics (BIG) of Chinese Academy of Sciences (CAS),2,0.68368,0.67882,0.0342,0.02328,0.95375,0.96623,0.962,0.9572,150,150,B,B,biological fallback assumption,illumina,hiseq_era,full_length,random_priming,smarter,bulk,clip,iclip,,China,2018-09-28,Blastula,Embryo,Whole Organism,All anatomical structures
49549,SRR7942636,SRX4776901,SRS3857441,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 in 200 ul PK buffer containing 20 ul proteinase K Roche  0311582001 for 1 h at 55°C. 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 TURBOâ„¢ 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/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 in 200 ul PK buffer containing 20 ul proteinase K Roche  0311582001 for 1 h at 55°C. 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°C in 10× RNA Fragmentation Reagent Ambion  then stopped by 10× RNA stop solution Ambion  and precipitated with 100% ethanol. The RNA pellet was resuspended in 100 µl bisulfite solution pH 5.1  which is a 100:1 mixture of 40% sodium bisulfite Sigma and 600 µM hydroquinone Sigma and subjected to heat incubation at 75°C 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 °C for 1 h. post ethanol precipitation  the RNA was resuspended in 11 µl 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,,,RIP 4hpf rep2,GSM3406902,,source name:Zebrafish embryo|strain:AB strain|cell type:Zebrafish embryo|age:4 hpf|tissue:whole embryo,RIP 4hpf rep2,RIP seq and iCLIP seq reads were aligned to the zv9 genome assembly using TopHat v2.1.1 and BWA v0.7.10  respectively. RIP seq: MACS2 v2.1.1 were used for the peak calling and peaks were annotated by applying BEDTools’ intersectBed. iCLIP: To identify the Ybx1 targets’ locus  the mode of truncation calling was performed. For each truncation position  the coverages of unique tag k and truncations m were determined by CIMS.pl program CLIP Tool Kit  CTK. The sites were annotated by applying BEDTools’ intersectBed. Genome build: zv9 Supplementary files format and content: Ybx1 binding sites in two biological replicates.,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 in 200 ul PK buffer containing 20 ul proteinase K Roche  0311582001 for 1 h at 55°C. 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™ 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.,,strain:AB strain|cell type:Zebrafish embryo|age:4 hpf|tissue:whole embryo,GSM3406902,GSM3406902: RIP 4hpf rep2; Danio rerio; RIP Seq,GSM3406902,,1,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 in 200 ul PK buffer containing 20 ul proteinase K Roche  0311582001 for 1 h at 55°C. 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™ 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.,GEO Accession:GSM3406902,RIP-Seq,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,Illumina HiSeq 2500,,SRP162876,,,RIP_4hpf_rep2_R1.fastq.gz RIP_4hpf_rep2_R2.fastq.gz,fastq fastq,22888305600.0,76294352.0,GSM3406902 r1,0:150 1:150,A:5927520807;C:4846894836;G:4996844576;T:7111789925;N:5255456,150,150,,,5927520807,4846894836,4996844576,7111789925,5255456,SRX4776901,SRS3857441,SRA786939,GEO,Beijing Institute of Genomics (BIG) of Chinese Academy of Sciences (CAS),2,0.2704,0.17205,0.06114,0.06961,0.95879,0.9698,0.94515,0.83252,150,150,B,B,mate2-mate1 similar by mapping diff,illumina,hiseq_era,full_length,random_priming,smarter,bulk,clip,iclip,,China,2018-09-28,Blastula,Embryo,Whole Organism,All anatomical structures
49550,SRR7942635,SRX4776900,SRS3857440,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 in 200 ul PK buffer containing 20 ul proteinase K Roche  0311582001 for 1 h at 55°C. 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 TURBOâ„¢ 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/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 in 200 ul PK buffer containing 20 ul proteinase K Roche  0311582001 for 1 h at 55°C. 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°C in 10× RNA Fragmentation Reagent Ambion  then stopped by 10× RNA stop solution Ambion  and precipitated with 100% ethanol. The RNA pellet was resuspended in 100 µl bisulfite solution pH 5.1  which is a 100:1 mixture of 40% sodium bisulfite Sigma and 600 µM hydroquinone Sigma and subjected to heat incubation at 75°C 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 °C for 1 h. post ethanol precipitation  the RNA was resuspended in 11 µl 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,,,RIP 4hpf rep1,GSM3406901,,source name:Zebrafish embryo|strain:AB strain|cell type:Zebrafish embryo|age:4 hpf|tissue:whole embryo,RIP 4hpf rep1,RIP seq and iCLIP seq reads were aligned to the zv9 genome assembly using TopHat v2.1.1 and BWA v0.7.10  respectively. RIP seq: MACS2 v2.1.1 were used for the peak calling and peaks were annotated by applying BEDTools’ intersectBed. iCLIP: To identify the Ybx1 targets’ locus  the mode of truncation calling was performed. For each truncation position  the coverages of unique tag k and truncations m were determined by CIMS.pl program CLIP Tool Kit  CTK. The sites were annotated by applying BEDTools’ intersectBed. Genome build: zv9 Supplementary files format and content: Ybx1 binding sites in two biological replicates.,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 in 200 ul PK buffer containing 20 ul proteinase K Roche  0311582001 for 1 h at 55°C. 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™ 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.,,strain:AB strain|cell type:Zebrafish embryo|age:4 hpf|tissue:whole embryo,GSM3406901,GSM3406901: RIP 4hpf rep1; Danio rerio; RIP Seq,GSM3406901,,1,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 in 200 ul PK buffer containing 20 ul proteinase K Roche  0311582001 for 1 h at 55°C. 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™ 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.,GEO Accession:GSM3406901,RIP-Seq,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,Illumina HiSeq 2500,,SRP162876,,,RIP_4hpf_rep1_R1.fastq.gz RIP_4hpf_rep1_R2.fastq.gz,fastq fastq,19929300600.0,66431002.0,GSM3406901 r1,0:150 1:150,A:5076575451;C:4235012507;G:4442151234;T:6170975339;N:4586069,150,150,,,5076575451,4235012507,4442151234,6170975339,4586069,SRX4776900,SRS3857440,SRA786939,GEO,Beijing Institute of Genomics (BIG) of Chinese Academy of Sciences (CAS),2,0.27932,0.16557,0.06585,0.06178,0.95692,0.96844,0.94238,0.84334,150,150,B,B,mate2-mate1 similar by mapping diff,illumina,hiseq_era,full_length,random_priming,smarter,bulk,clip,iclip,,China,2018-09-28,Blastula,Embryo,Whole Organism,All anatomical structures
49573,SRR10434662,SRX7130632,SRS5639976,SRP163087,PRJNA494251,Dynamic landscapes and regulome of mRNA structure during zebrafish early embryogenesis,GSE120724,Other,We resolved the RNA secondary structure during zebrafish early embryogenesis based on  in vivo click selective 2' hydroxyl acylation and profiling experiment icSHAPE. We analyzed the RNA structure dynamics among different development stages.  Also  we studied which factors regulate maternal gene decay by RNA structure switch. Overall design: icSHAPE of 12 samples from zebrafish embryos at six different early development stages and 4 samples from Elavl1a deficient zebrafish embryos at sphere and shield stages  including two treated with DMSO as control and two treated with NAIN3 for each stage or condition. RNA seq of 6 samples from zebrafish embryos at 2  4  6 h.p.f. and 4 samples from Elavl1a deficient zebrafish embryos at 4 and 6 h.p.f. including two biological replicates. iCLIP seq of 4 samples from flag elavl1a mRNA injected zebrafish embryos at xxx and 6 h.p.f. including two biological replicates.,,pubmed:32423473,,shield Flag Elavl1a iCLIP rep2,GSM4157939,,tissue:zebrafish embryos|strain:zebrafish embryos|cell type:normal zebrafish embryos cells|age:6hpf,shield Flag Elavl1a iCLIP rep2,"The proceesing procedure is similar to CTK toolhttps://zhanglab.c2b2.columbia.edu/index.php/ICLIP data analysis using CTK. First  we trimmed three prime adaptor from reads by cutadapt collapsed duplicated reads by fastq2collapse.pl in CTK tool stripped  5’ degenerated barcode mapped clean reads to zebrafish reference genomez10 by bwav0.7.17. data from replicates was merged for peak calling We use CTK tool CITS mode to call the peaks. Reads were clustered using tag2cluster.pl with parameters  big  s  maxgap "" 1"" peaks are identified using tag2peak.pl with parameters  big  ss  v   prefix ""CITS""  gap 25  p 0.05 Genome build: z10 Supplementary files format and content: iCLIP peaks identified by CTK tool were provided in bed file format. The first three columns represent the 0 based location of the peak in the genome and the last column represents the strand.The fourth column reprents the peak ID and describes the gene locus  peak heightPH  expected PH/backgroundPH0 and pvalueP. The fifth  column represents the peak height.",zebrafish embryos,,iCLIP seq was carried out as previously described Despic et al.  2017. Breifly  flag elavl1a mRNA injected embryos were collected at xxx h.p.f. and 6 h.p.f.. 400 zebrafish embryos were irradiated twice with 0.8 J/cm2 Stratalinker 2400  Stratagene  lysed  and subjected to mild RNA fragmentation. Crosslinked RNA proteins complexes were immunopurified using Anti FLAG M2 Magnetic Beads Merck  M8823 for 4 h.p.f. and 6 h.p.f. at 4 °C. RNA was extracted Library construction was performed by using Smarter smRNA Seq kit Clontech Laboratories Inc,Zebrafish wild type strain AB was raised in system water at 28.5°C under standard conditions.,strain:zebrafish embryos|cell type:normal zebrafish embryos cells|age:6hpf,GSM4157939,GSM4157939: shield Flag Elavl1a iCLIP rep2; Danio rerio; OTHER,GSM4157939,,1,iCLIP seq was carried out as previously described Despic et al.  2017. Breifly  flag elavl1a mRNA injected embryos were collected at xxx h.p.f. and 6 h.p.f.. 400 zebrafish embryos were irradiated twice with 0.8 J/cm2 Stratalinker 2400  Stratagene  lysed  and subjected to mild RNA fragmentation. Crosslinked RNA proteins complexes were immunopurified using Anti FLAG M2 Magnetic Beads Merck  M8823 for 4 h.p.f. and 6 h.p.f. at 4 °C. RNA was extracted Library construction was performed by using Smarter smRNA Seq kit Clontech Laboratories Inc,GEO Accession:GSM4157939,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,HiSeq X Ten,,SRP163087,,,Shield_Elavl1a_iCLIP_rep2.R2.fq.gz Shield_Elavl1a_iCLIP_rep2.R1.fq.gz,fastq fastq,23879342400.0,79597808.0,GSM4157939 r1,0:150 1:150,A:6335254303;C:5166953187;G:6017120995;T:6358132998;N:1880917,150,150,,,6335254303,5166953187,6017120995,6358132998,1880917,SRX7130632,SRS5639976,SRA787572,GEO,"Life Science, Tsinghua University",2,0.14294,0.15151,0.03583,0.08959,0.99397,0.98269,0.96727,0.75507,150,150,B,B,biological fallback assumption,illumina,hiseq_era,full_length,poly_a,smarter,bulk,clip,iclip,,China,2019-11-12,Gastrula,Embryo,Embryo Imprecise,All anatomical structures
49574,SRR10434661,SRX7130631,SRS5639975,SRP163087,PRJNA494251,Dynamic landscapes and regulome of mRNA structure during zebrafish early embryogenesis,GSE120724,Other,We resolved the RNA secondary structure during zebrafish early embryogenesis based on  in vivo click selective 2' hydroxyl acylation and profiling experiment icSHAPE. We analyzed the RNA structure dynamics among different development stages.  Also  we studied which factors regulate maternal gene decay by RNA structure switch. Overall design: icSHAPE of 12 samples from zebrafish embryos at six different early development stages and 4 samples from Elavl1a deficient zebrafish embryos at sphere and shield stages  including two treated with DMSO as control and two treated with NAIN3 for each stage or condition. RNA seq of 6 samples from zebrafish embryos at 2  4  6 h.p.f. and 4 samples from Elavl1a deficient zebrafish embryos at 4 and 6 h.p.f. including two biological replicates. iCLIP seq of 4 samples from flag elavl1a mRNA injected zebrafish embryos at xxx and 6 h.p.f. including two biological replicates.,,pubmed:32423473,,shield Flag Elavl1a iCLIP rep1,GSM4157938,,tissue:zebrafish embryos|strain:zebrafish embryos|cell type:normal zebrafish embryos cells|age:6hpf,shield Flag Elavl1a iCLIP rep1,"The proceesing procedure is similar to CTK toolhttps://zhanglab.c2b2.columbia.edu/index.php/ICLIP data analysis using CTK. First  we trimmed three prime adaptor from reads by cutadapt collapsed duplicated reads by fastq2collapse.pl in CTK tool stripped  5’ degenerated barcode mapped clean reads to zebrafish reference genomez10 by bwav0.7.17. data from replicates was merged for peak calling We use CTK tool CITS mode to call the peaks. Reads were clustered using tag2cluster.pl with parameters  big  s  maxgap "" 1"" peaks are identified using tag2peak.pl with parameters  big  ss  v   prefix ""CITS""  gap 25  p 0.05 Genome build: z10 Supplementary files format and content: iCLIP peaks identified by CTK tool were provided in bed file format. The first three columns represent the 0 based location of the peak in the genome and the last column represents the strand.The fourth column reprents the peak ID and describes the gene locus  peak heightPH  expected PH/backgroundPH0 and pvalueP. The fifth  column represents the peak height.",zebrafish embryos,,iCLIP seq was carried out as previously described Despic et al.  2017. Breifly  flag elavl1a mRNA injected embryos were collected at xxx h.p.f. and 6 h.p.f.. 400 zebrafish embryos were irradiated twice with 0.8 J/cm2 Stratalinker 2400  Stratagene  lysed  and subjected to mild RNA fragmentation. Crosslinked RNA proteins complexes were immunopurified using Anti FLAG M2 Magnetic Beads Merck  M8823 for 4 h.p.f. and 6 h.p.f. at 4 °C. RNA was extracted Library construction was performed by using Smarter smRNA Seq kit Clontech Laboratories Inc,Zebrafish wild type strain AB was raised in system water at 28.5°C under standard conditions.,strain:zebrafish embryos|cell type:normal zebrafish embryos cells|age:6hpf,GSM4157938,GSM4157938: shield Flag Elavl1a iCLIP rep1; Danio rerio; OTHER,GSM4157938,,1,iCLIP seq was carried out as previously described Despic et al.  2017. Breifly  flag elavl1a mRNA injected embryos were collected at xxx h.p.f. and 6 h.p.f.. 400 zebrafish embryos were irradiated twice with 0.8 J/cm2 Stratalinker 2400  Stratagene  lysed  and subjected to mild RNA fragmentation. Crosslinked RNA proteins complexes were immunopurified using Anti FLAG M2 Magnetic Beads Merck  M8823 for 4 h.p.f. and 6 h.p.f. at 4 °C. RNA was extracted Library construction was performed by using Smarter smRNA Seq kit Clontech Laboratories Inc,GEO Accession:GSM4157938,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,HiSeq X Ten,,SRP163087,,,Shield_Elavl1a_iCLIP_rep1.R1.fq.gz Shield_Elavl1a_iCLIP_rep1.R2.fq.gz,fastq fastq,22375977600.0,74586592.0,GSM4157938 r1,0:150 1:150,A:6243025236;C:5008499337;G:5418170547;T:5704514909;N:1767571,150,150,,,6243025236,5008499337,5418170547,5704514909,1767571,SRX7130631,SRS5639975,SRA787572,GEO,"Life Science, Tsinghua University",2,0.05144,0.12625,0.01219,0.09577,0.99734,0.98113,0.8629,0.52429,150,150,B,B,mate1-mate2 similar by mapping diff,illumina,hiseq_era,full_length,poly_a,smarter,bulk,clip,iclip,,China,2019-11-12,Gastrula,Embryo,Embryo Imprecise,All anatomical structures
49575,SRR10434660,SRX7130630,SRS5639974,SRP163087,PRJNA494251,Dynamic landscapes and regulome of mRNA structure during zebrafish early embryogenesis,GSE120724,Other,We resolved the RNA secondary structure during zebrafish early embryogenesis based on  in vivo click selective 2' hydroxyl acylation and profiling experiment icSHAPE. We analyzed the RNA structure dynamics among different development stages.  Also  we studied which factors regulate maternal gene decay by RNA structure switch. Overall design: icSHAPE of 12 samples from zebrafish embryos at six different early development stages and 4 samples from Elavl1a deficient zebrafish embryos at sphere and shield stages  including two treated with DMSO as control and two treated with NAIN3 for each stage or condition. RNA seq of 6 samples from zebrafish embryos at 2  4  6 h.p.f. and 4 samples from Elavl1a deficient zebrafish embryos at 4 and 6 h.p.f. including two biological replicates. iCLIP seq of 4 samples from flag elavl1a mRNA injected zebrafish embryos at xxx and 6 h.p.f. including two biological replicates.,,pubmed:32423473,,sphere Flag Elavl1a iCLIP rep2,GSM4157937,,tissue:zebrafish embryos|strain:zebrafish embryos|cell type:normal zebrafish embryos cells|age:4hpf,sphere Flag Elavl1a iCLIP rep2,"The proceesing procedure is similar to CTK toolhttps://zhanglab.c2b2.columbia.edu/index.php/ICLIP data analysis using CTK. First  we trimmed three prime adaptor from reads by cutadapt collapsed duplicated reads by fastq2collapse.pl in CTK tool stripped  5’ degenerated barcode mapped clean reads to zebrafish reference genomez10 by bwav0.7.17. data from replicates was merged for peak calling We use CTK tool CITS mode to call the peaks. Reads were clustered using tag2cluster.pl with parameters  big  s  maxgap "" 1"" peaks are identified using tag2peak.pl with parameters  big  ss  v   prefix ""CITS""  gap 25  p 0.05 Genome build: z10 Supplementary files format and content: iCLIP peaks identified by CTK tool were provided in bed file format. The first three columns represent the 0 based location of the peak in the genome and the last column represents the strand.The fourth column reprents the peak ID and describes the gene locus  peak heightPH  expected PH/backgroundPH0 and pvalueP. The fifth  column represents the peak height.",zebrafish embryos,,iCLIP seq was carried out as previously described Despic et al.  2017. Breifly  flag elavl1a mRNA injected embryos were collected at xxx h.p.f. and 6 h.p.f.. 400 zebrafish embryos were irradiated twice with 0.8 J/cm2 Stratalinker 2400  Stratagene  lysed  and subjected to mild RNA fragmentation. Crosslinked RNA proteins complexes were immunopurified using Anti FLAG M2 Magnetic Beads Merck  M8823 for 4 h.p.f. and 6 h.p.f. at 4 °C. RNA was extracted Library construction was performed by using Smarter smRNA Seq kit Clontech Laboratories Inc,Zebrafish wild type strain AB was raised in system water at 28.5°C under standard conditions.,strain:zebrafish embryos|cell type:normal zebrafish embryos cells|age:4hpf,GSM4157937,GSM4157937: sphere Flag Elavl1a iCLIP rep2; Danio rerio; OTHER,GSM4157937,,1,iCLIP seq was carried out as previously described Despic et al.  2017. Breifly  flag elavl1a mRNA injected embryos were collected at xxx h.p.f. and 6 h.p.f.. 400 zebrafish embryos were irradiated twice with 0.8 J/cm2 Stratalinker 2400  Stratagene  lysed  and subjected to mild RNA fragmentation. Crosslinked RNA proteins complexes were immunopurified using Anti FLAG M2 Magnetic Beads Merck  M8823 for 4 h.p.f. and 6 h.p.f. at 4 °C. RNA was extracted Library construction was performed by using Smarter smRNA Seq kit Clontech Laboratories Inc,GEO Accession:GSM4157937,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,HiSeq X Ten,,SRP163087,,,Sphere_Elavl1a_iCLIP_rep2.R1.fq.gz Sphere_Elavl1a_iCLIP_rep2.R2.fq.gz,fastq fastq,22090855200.0,73636184.0,GSM4157937 r1,0:150 1:150,A:5631298996;C:5114935575;G:5742247735;T:5600647498;N:1725396,150,150,,,5631298996,5114935575,5742247735,5600647498,1725396,SRX7130630,SRS5639974,SRA787572,GEO,"Life Science, Tsinghua University",2,0.23476,0.17586,0.06861,0.08859,0.99563,0.98873,0.95465,0.79994,150,150,B,B,mate2-mate1 similar by mapping diff,illumina,hiseq_era,full_length,poly_a,smarter,bulk,clip,iclip,,China,2019-11-12,Blastula,Embryo,Embryo Imprecise,All anatomical structures
49576,SRR10434659,SRX7130629,SRS5639973,SRP163087,PRJNA494251,Dynamic landscapes and regulome of mRNA structure during zebrafish early embryogenesis,GSE120724,Other,We resolved the RNA secondary structure during zebrafish early embryogenesis based on  in vivo click selective 2' hydroxyl acylation and profiling experiment icSHAPE. We analyzed the RNA structure dynamics among different development stages.  Also  we studied which factors regulate maternal gene decay by RNA structure switch. Overall design: icSHAPE of 12 samples from zebrafish embryos at six different early development stages and 4 samples from Elavl1a deficient zebrafish embryos at sphere and shield stages  including two treated with DMSO as control and two treated with NAIN3 for each stage or condition. RNA seq of 6 samples from zebrafish embryos at 2  4  6 h.p.f. and 4 samples from Elavl1a deficient zebrafish embryos at 4 and 6 h.p.f. including two biological replicates. iCLIP seq of 4 samples from flag elavl1a mRNA injected zebrafish embryos at xxx and 6 h.p.f. including two biological replicates.,,pubmed:32423473,,sphere Flag Elavl1a iCLIP rep1,GSM4157936,,tissue:zebrafish embryos|strain:zebrafish embryos|cell type:normal zebrafish embryos cells|age:4hpf,sphere Flag Elavl1a iCLIP rep1,"The proceesing procedure is similar to CTK toolhttps://zhanglab.c2b2.columbia.edu/index.php/ICLIP data analysis using CTK. First  we trimmed three prime adaptor from reads by cutadapt collapsed duplicated reads by fastq2collapse.pl in CTK tool stripped  5’ degenerated barcode mapped clean reads to zebrafish reference genomez10 by bwav0.7.17. data from replicates was merged for peak calling We use CTK tool CITS mode to call the peaks. Reads were clustered using tag2cluster.pl with parameters  big  s  maxgap "" 1"" peaks are identified using tag2peak.pl with parameters  big  ss  v   prefix ""CITS""  gap 25  p 0.05 Genome build: z10 Supplementary files format and content: iCLIP peaks identified by CTK tool were provided in bed file format. The first three columns represent the 0 based location of the peak in the genome and the last column represents the strand.The fourth column reprents the peak ID and describes the gene locus  peak heightPH  expected PH/backgroundPH0 and pvalueP. The fifth  column represents the peak height.",zebrafish embryos,,iCLIP seq was carried out as previously described Despic et al.  2017. Breifly  flag elavl1a mRNA injected embryos were collected at xxx h.p.f. and 6 h.p.f.. 400 zebrafish embryos were irradiated twice with 0.8 J/cm2 Stratalinker 2400  Stratagene  lysed  and subjected to mild RNA fragmentation. Crosslinked RNA proteins complexes were immunopurified using Anti FLAG M2 Magnetic Beads Merck  M8823 for 4 h.p.f. and 6 h.p.f. at 4 °C. RNA was extracted Library construction was performed by using Smarter smRNA Seq kit Clontech Laboratories Inc,Zebrafish wild type strain AB was raised in system water at 28.5°C under standard conditions.,strain:zebrafish embryos|cell type:normal zebrafish embryos cells|age:4hpf,GSM4157936,GSM4157936: sphere Flag Elavl1a iCLIP rep1; Danio rerio; OTHER,GSM4157936,,1,iCLIP seq was carried out as previously described Despic et al.  2017. Breifly  flag elavl1a mRNA injected embryos were collected at xxx h.p.f. and 6 h.p.f.. 400 zebrafish embryos were irradiated twice with 0.8 J/cm2 Stratalinker 2400  Stratagene  lysed  and subjected to mild RNA fragmentation. Crosslinked RNA proteins complexes were immunopurified using Anti FLAG M2 Magnetic Beads Merck  M8823 for 4 h.p.f. and 6 h.p.f. at 4 °C. RNA was extracted Library construction was performed by using Smarter smRNA Seq kit Clontech Laboratories Inc,GEO Accession:GSM4157936,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,HiSeq X Ten,,SRP163087,,,Sphere_Elavl1a_iCLIP_rep1.R2.fq.gz Sphere_Elavl1a_iCLIP_rep1.R1.fq.gz,fastq fastq,15794202300.0,52647341.0,GSM4157936 r1,0:150 1:150,A:4443931911;C:3527112429;G:3707720167;T:4114188890;N:1248903,150,150,,,4443931911,3527112429,3707720167,4114188890,1248903,SRX7130629,SRS5639973,SRA787572,GEO,"Life Science, Tsinghua University",2,0.06877,0.10388,0.02139,0.07135,0.99776,0.98413,0.87145,0.57456,150,150,B,B,mate1-mate2 similar by mapping diff,illumina,hiseq_era,full_length,poly_a,smarter,bulk,clip,iclip,,China,2019-11-12,Blastula,Embryo,Embryo Imprecise,All anatomical structures