{"database": "metadata", "table": "run_metadata", "rows": [[72704, "SRR23071972", "SRX19025110", "SRS16444159", "SRP417272", "PRJNA923391", "MBD5  a m5C RNA reader  regulates histone ubiquitylation in development and function", "GSE222803", "Other", "Methyl CpG binding domain MBD containing proteins are readers of epigenetic information. The founding member  MeCP2  mutated in the Rett Syndrome  binds to methylated CpG DNAs.  MBD5 is an evolutionarily conserved MBD family member associated with epilepsy  autism spectrum disorders ASD  and early onset dementia. The mechanism of action of MBD5 remains unclear. Here we report that mbd5 regulates embryonic development  erythrocyte differentiation  iron metabolism  and behavior in zebrafish. MBD5 is essential to activate the expression of erythrocyte differentiation genes e.g.  hbae3  iron regulated genes e.g.  fth1  and ASD related genes e.g.  gabbr2  by binding to 5 methylcytosine m5C modified mRNAs  interacting with and stabilizing Additional Sex Combs Like 1 ASXL1  a component of the Polycomb Repressive Deubiquinase PR DUB complex  to facilitate removal of the repressive monoubiquitin mark at histone H2A H2A K119Ub  thereby facilitating gene activation. Together  these findings reveal MBD5 as a m5C RNA reader that links RNA methylation to histone modification in vivo. Overall design: [Dataset 1] 6 samples. Triplicates for ribo minus RNA Seq from 78 hpf zebrafish embryos treated with control or mbd5 morpholino [Dataset 2] 16 samples. Duplicates for CLIP seq experiments from 48 hpf 78 hpf zebrafish embryos treated with 0.5 mM DFO or 1 mM FAC", null, "pubmed:38366571", null, "ctrl input rep1", "GSM6932544", null, "source name:whole embryo|tissue:whole embryo|genotype:Tg[zhsp70l:FLAGmbd5iso2 E2AGFP]|treatment:No treatment|geo loc name:missing|collection date:missing", "ctrl input rep1", "Adapter trimming by Cutadapt Reads mapping by STAR to the transcriptome peak calling with pureCLIP or differential gene expression with DESeq2 Assembly: dr10 Supplementary files format and content: For [Dataset 1]  excel xlsx files  with differential expression by DESeq2. For [Dataset 2]  excel xlsx files  with CLIP seq peaks identified by pureCLIP algorithm", "whole embryo", "For [Dataset 1]  morpholino oligonucleotides are applied by microinjection. For [Dataset 2]  FAC F5879  Sigma Aldrich was dissolved in E3 medium at 50 mM and stored at 4\u00b0C. zebrafish were treated with 1 mM fresh FAC from 3 dpf 6 dpf every day prior to behavior analysis. DFO D9533  Sigma Aldrich was dissolved in E3 medium at 150 mM and stored at  20\u00b0C. Zebrafish were treated with 0.5 mM DFO from 3 dpf 6 dpf  replenished every day  prior to behavior analysis.", "For [Dataset 1]  total RNAs were extracted with standard TRIzol protocol. For [Dataset 2]  76 hpf larval zebrafish were UV crosslinked and flash frozen in liquid nitrogen. Pellets were thawed on ice and resuspended in 3 volume of ice cold CLIP lysis buffer 50 mM HEPES pH 7.5  150 mM KCl  2 mM EDTA  0.5% v/v NP 40  0.5 mM DTT  1 \u00d7 Halt\u2122 Protease and Phosphatase Inhibitor Cocktail  1 \u00d7 RNaseOUT Recombinant Ribonuclease Inhibitor. Pellets were lysed by rotating at 4 \u00b0C for 15 minutes post passing through a 26 G needle BD Biosciences. Embryo suspensions were sonicated on a bioruptor Diagenode with 30 s on/30 s off for 5 cycles. Lysates were cleared by centrifugation at 21 000 g for 15 minutes at 4 \u00b0C on a benchtop centrifuge. Supernatants were applied to Flag antibody Abcam conjugated protein A beads Invitrogen and left overnight at 4 \u00b0C on an end to end rotor. Beads were washed extensively with 1 ml wash buffer 50 mM HEPES pH 7.5  300 mM KCl  0.05% v/v NP 40  1 \u00d7 Halt\u2122 Protease and Phosphatase Inhibitor Cocktail  1 \u00d7 RNaseOUT Recombinant Ribonuclease Inhibitor at 4 \u00b0C for 5 times. Protein RNA complex conjugated to the beads were treated by 8 U/\u03bcL RNase T1 Thermo Scientific at 22 \u00b0C for 10 minutes with shaking. Input samples are digested in parallel. Then input and IP samples were separated on an SDS PAGE gel and gel slices at corresponding size ranges were treated by proteinase K Invitrogen elution. RNA was recovered with TRIZol reagent Invitrogen. Then T4 PNK Thermo Scientific end repair was performed with purified RNA before library constructions. For [Dataset 1]  RNA libraries for input samples were constructed by SMARTer Stranded Total RNA Seq Kit v2 TaKaRa  634417 with ribo minus RNA as input.  For [Dataset 2]  RNA libraries were constructed with NEB small RNA kit NEB. Libraries were pooled and sequenced on a NovaSeq 6000 sequencer with paired end mode 50 bp setting.", "Zebrafish embryos were treated with 0.003% 1 phenyl 2 thiourea PTU in E3 medium to prevent pigment formation. 3 month 9 month aged wild type or mbd5 mutant lines generated in this study were used for breeding or adult behavior analyses. Developmental stages were determined according to their morphology.", "tissue:whole embryo|genotype:Tg[zhsp70l:FLAGmbd5iso2 E2AGFP]|treatment:No treatment", "GSM6932544", "GSM6932544: ctrl input rep1; Danio rerio; RIP Seq", "GSM6932544 r1", "GSM6932544", "1", "For [Dataset 1]  total RNAs were extracted with standard TRIzol protocol. For [Dataset 2]  76 hpf larval zebrafish were UV crosslinked and flash frozen in liquid nitrogen. Pellets were thawed on ice and resuspended in 3 volume of ice cold CLIP lysis buffer 50 mM HEPES pH 7.5  150 mM KCl  2 mM EDTA  0.5% v/v NP 40  0.5 mM DTT  1 \u00d7 Halt\u2122 Protease and Phosphatase Inhibitor Cocktail  1 \u00d7 RNaseOUT Recombinant Ribonuclease Inhibitor. Pellets were lysed by rotating at 4 \u00b0C for 15 minutes post passing through a 26 G needle BD Biosciences. Embryo suspensions were sonicated on a bioruptor Diagenode with 30 s on/30 s off for 5 cycles. Lysates were cleared by centrifugation at 21 000 g for 15 minutes at 4 \u00b0C on a benchtop centrifuge. Supernatants were applied to Flag antibody Abcam conjugated protein A beads Invitrogen and left overnight at 4 \u00b0C on an end to end rotor. Beads were washed extensively with 1 ml wash buffer 50 mM HEPES pH 7.5  300 mM KCl  0.05% v/v NP 40  1 \u00d7 Halt\u2122 Protease and Phosphatase Inhibitor Cocktail  1 \u00d7 RNaseOUT Recombinant Ribonuclease Inhibitor at 4 \u00b0C for 5 times. Protein RNA complex conjugated to the beads were treated by 8 U/\u03bcL RNase T1 Thermo Scientific at 22 \u00b0C for 10 minutes with shaking. Input samples are digested in parallel. Then input and IP samples were separated on an SDS PAGE gel and gel slices at corresponding size ranges were treated by proteinase K Invitrogen elution. RNA was recovered with TRIZol reagent Invitrogen. Then T4 PNK Thermo Scientific end repair was performed with purified RNA before library constructions. For [Dataset 1]  RNA libraries for input samples were constructed by SMARTer Stranded Total RNA Seq Kit v2 TaKaRa  634417 with ribo minus RNA as input.  For [Dataset 2]  RNA libraries were constructed with NEB small RNA kit NEB. Libraries were pooled and sequenced on a NovaSeq 6000 sequencer with paired end mode 50 bp setting.", null, "RIP-Seq", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP417272", null, "loader:fastq load.py", "ctrl_input_rep1.r1.fastq.gz ctrl_input_rep1.r2.fastq.gz", "fastq fastq", 2870115000.0, 23917625.0, "GSM6932544 r1", "0:60 1:60", "A:616586993;C:849261640;G:916690149;T:487568187;N:8031", 60, 60, null, null, 616586993, 849261640, 916690149, 487568187, 8031, "SRX19025110", "SRS16444159", "SRA1573941", "University of Chicago", "University of Chicago", 2, 0.76321, 0.76237, 0.06602, 0.06566, 0.97064, 0.96903, 0.68553, 0.75314, 60, 60, "B", "B", "biological fallback assumption", "illumina", "novaseq_era", "full_length", "small_rna", "smarter", "bulk", "unknown", "unknown", null, "United States", "2023-01-12", "Multi-stage", "Multi-stage", "Whole Organism", "All anatomical structures"]], "columns": ["rowid", "run.accession", "experiment.accession", "sample.accession", "study.accession", "bioproject", "study.title", "study.alias", "study.type", "study.abstract", "study.attributes", "study.PMIDs", "sample.description", "sample.title", "sample.alias", "sample.centername", "sample.attributes", "GEOsample.title", "GEOsample.dataprocessing", "GEOsample.source", "GEOsample.treatmentprotocol", "GEOsample.extractprotocol", "GEOsample.growthprotocol", "GEOsample.characteristics", "GEOsample.accession", "experiment.title", "experiment.alias", "experiment.library_name", "experiment.design_description", "experiment.library_construction_protocol", "experiment.attributes", "experiment.library_strategy", "experiment.library_source", "experiment.library_selection", "experiment.library_layout", "experiment.platform", "experiment.instrument_model", "experiment.spot_descriptor", "experiment.study_ref", "run.title", "run.attributes", "run.filename", "run.semantic_name", "run.total_bases", "run.total_spots", "run.alias", "run.read_lengths", "run.base_counts", "run.r1_length", "run.r2_length", "run.r3_length", "run.r4_length", "run.Acount", "run.Ccount", "run.Gcount", "run.Tcount", "run.Ncount", "run.experiment", "run.pool_member", "submission.accession", "submission.srasource", "submission.bioprojectsource", "seqdetective.n_mates", "seqdetective.mapping_rate.mate1", "seqdetective.mapping_rate.mate2", "seqdetective.nofeature_rate.mate1", "seqdetective.nofeature_rate.mate2", "seqdetective.sparsity.mate1", "seqdetective.sparsity.mate2", "seqdetective.pos_strand_rate.mate1", "seqdetective.pos_strand_rate.mate2", "seqdetective.readlen.mate1", "seqdetective.readlen.mate2", "seqdetective.judgement.mate1", "seqdetective.judgement.mate2", "seqdetective.judgement.reason", "platform_family", "instrument_generation", "read_bias", "selection_class", "prep_kit", "sc_or_bulk", "tech_class", "technology", "tech_variant", "submission.bioprojectsource.country", "earliest_date", "devstage_curation", "devstage_curation_coarse", "tissue_curation", "tissue_curation_coarse"], "primary_keys": ["rowid"], "primary_key_values": ["72704"], "units": {}, "query_ms": 24.909919004130643}