run_metadata: 72705

This data as json

rowid	run.accession	experiment.accession	sample.accession	study.accession	bioproject	study.title	study.alias	study.type	study.abstract	study.attributes	study.PMIDs	sample.description	sample.title	sample.alias	sample.centername	sample.attributes	GEOsample.title	GEOsample.dataprocessing	GEOsample.source	GEOsample.treatmentprotocol	GEOsample.extractprotocol	GEOsample.growthprotocol	GEOsample.characteristics	GEOsample.accession	experiment.title	experiment.alias	experiment.library_name	experiment.design_description	experiment.library_construction_protocol	experiment.attributes	experiment.library_strategy	experiment.library_source	experiment.library_selection	experiment.library_layout	experiment.platform	experiment.instrument_model	experiment.spot_descriptor	experiment.study_ref	run.title	run.attributes	run.filename	run.semantic_name	run.total_bases	run.total_spots	run.alias	run.read_lengths	run.base_counts	run.r1_length	run.r2_length	run.r3_length	run.r4_length	run.Acount	run.Ccount	run.Gcount	run.Tcount	run.Ncount	run.experiment	run.pool_member	submission.accession	submission.srasource	submission.bioprojectsource	seqdetective.n_mates	seqdetective.mapping_rate.mate1	seqdetective.mapping_rate.mate2	seqdetective.nofeature_rate.mate1	seqdetective.nofeature_rate.mate2	seqdetective.sparsity.mate1	seqdetective.sparsity.mate2	seqdetective.pos_strand_rate.mate1	seqdetective.pos_strand_rate.mate2	seqdetective.readlen.mate1	seqdetective.readlen.mate2	seqdetective.judgement.mate1	seqdetective.judgement.mate2	seqdetective.judgement.reason	platform_family	instrument_generation	read_bias	selection_class	prep_kit	sc_or_bulk	tech_class	technology	tech_variant	submission.bioprojectsource.country	earliest_date	devstage_curation	devstage_curation_coarse	tissue_curation	tissue_curation_coarse
72705	SRR23071973	SRX19025109	SRS16444158	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		pubmed:38366571		ctrl clip rep2	GSM6932543		source name:whole embryo\|tissue:whole embryo\|genotype:Tg[zhsp70l:FLAGmbd5iso2 E2AGFP]\|treatment:No treatment\|geo loc name:missing\|collection date:missing	ctrl clip rep2	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°C. 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°C. 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 × Halt™ Protease and Phosphatase Inhibitor Cocktail 1 × RNaseOUT Recombinant Ribonuclease Inhibitor. Pellets were lysed by rotating at 4 °C 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 °C on a benchtop centrifuge. Supernatants were applied to Flag antibody Abcam conjugated protein A beads Invitrogen and left overnight at 4 °C 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 × Halt™ Protease and Phosphatase Inhibitor Cocktail 1 × RNaseOUT Recombinant Ribonuclease Inhibitor at 4 °C for 5 times. Protein RNA complex conjugated to the beads were treated by 8 U/μL RNase T1 Thermo Scientific at 22 °C 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	GSM6932543	GSM6932543: ctrl clip rep2; Danio rerio; RIP Seq	GSM6932543 r1	GSM6932543	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 × Halt™ Protease and Phosphatase Inhibitor Cocktail 1 × RNaseOUT Recombinant Ribonuclease Inhibitor. Pellets were lysed by rotating at 4 °C 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 °C on a benchtop centrifuge. Supernatants were applied to Flag antibody Abcam conjugated protein A beads Invitrogen and left overnight at 4 °C 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 × Halt™ Protease and Phosphatase Inhibitor Cocktail 1 × RNaseOUT Recombinant Ribonuclease Inhibitor at 4 °C for 5 times. Protein RNA complex conjugated to the beads were treated by 8 U/μL RNase T1 Thermo Scientific at 22 °C 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.		RIP-Seq	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	Illumina NovaSeq 6000		SRP417272		loader:fastq load.py	ctrl_clip_rep2.r1.fastq.gz ctrl_clip_rep2.r2.fastq.gz	fastq fastq	3309978960.0	27583158.0	GSM6932543 r1	0:60 1:60	A:774105538;C:895792317;G:931799487;T:708272623;N:8995	60	60			774105538	895792317	931799487	708272623	8995	SRX19025109	SRS16444158	SRA1573941	University of Chicago	University of Chicago	2	0.94553	0.92262	0.21092	0.22735	0.90985	0.91407	0.67372	0.67171	60	60	B	B	biological fallback assumption	illumina	novaseq_era	full_length	small_rna	smarter	bulk	unknown	unknown		United States	2023-01-12	Multi-stage	Multi-stage	Whole Organism	All anatomical structures