run_metadata: 64447

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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
64447	SRR14703445	SRX11041466	SRS9110394	SRP322195	PRJNA734348	Methylome inheritance and enhancer dememorization construct an epigenetic gate safeguarding embryonic programs	GSE175951	Other	Unlike that of mammals the total DNA methylome of many cold blooded vertebrates is globally inherited from gametes to early embryos. In zebrafish this is however accompanied by sweeping “dememorization” of enhancers prior to fertilization for sperm and just post fertilization for oocyte as they undergo full methylation and are not demethylated again until phylotypic stage. The significance of both global methylome inheritance and enhancer dememorization in early embryos remains largely unknown. Adding to the puzzles the zygotic mutant zebrafish of dnmt1 the major DNA methylation maintenance methyltransferase surprisingly can develop to term. To solve the role of DNA methylation in early development we generated zebrafish embryos derived from dnmt1 knocking down oocytes using a recently developed method OMIS microinjection in situ which successfully eliminated DNA methylation before zygotic genome activation. dnmt1 deficient embryos failed to initiate epiboly and died around gastrulation. This is in part caused by activation of immune response and p53 regulated apoptosis likely triggered by the derepression of transposable elements. Single cell RNA seq further revealed defective differentiation in these mutants. DNA methylation is also required for the establishment of repressive histone marks H3K27me3 and H2AK119ub. Strikingly the loss of DNA methylation leads to extensive derepression of somatic genes and enhancers which acquire ectopic H3K27ac accessible chromatin and H3K4me3. These somatic enhancers are preferentially CG rich and are bound by CG containing TFs. By contrast embryonic enhancers are generally CG poor methylation insensitive and are bound by CG less TFs. Hence the global DNA methylome inheritance is essential for vertebrate early development and enhancer dememorization resets an epigenetic gate that separates embryonic and somatic programs. Overall design: By employing MethylC seq total RNA seq scRNA seq CUT&RUN ChIP seq and ATAC seq in control and dnmt1 mKD embryos at oocyte 256 cell dome and shield stages in zebrafish we interrogated the function of DNA methylome and its inheritance in early zebrafish development.		pubmed:34936444		5dpf tail dnmt1 zKO total RNA seq rep2	GSM5351821		source name:larva\|strain:TL\|genotype:dnmt1 / \|developmental stage:5dpf\|tissue:larva tail\|treatment:untreated	5dpf tail dnmt1 zKO total RNA seq rep2	All STEM seq datasets were mapped to the danRer7 reference genome by Bismark Krueger and Andrews 2011. Reads were trimmed with cutadaptor Martin 2011 using parameters: minimum length 20 pair filter=any. Alignments were performed with the following parameters: N 1 X 600 score min L 0 0.6. Multi mapped reads and PCR duplicates were removed. bismark methylation extractor was used to calculate the DNA methylation level. Total RNA seq data were firstly processed using Trim Galore! with default parameters to trim the adapter containing and low quality reads. The filtered data were then mapped to the zebrafish reference genome danRer7 by STAR version: STAR 2.5.3a modified Dobin et al. 2013 with the following parameters: outSAMstrandField intronMotif outSAMattributes All outSAMunmapped Within outSAMattrIHstart 0 outWigStrand Stranded outFilterMultimapNmax 20 twopassMode Basic. The gene expression level was normalized to fragments per kilobase of transcript per million mapped FPKM values using Cufflinks version 2.2.1 Trapnell et al. 2012. The scRNA seq data were processed with Cell Ranger 3.1 for genome alignment danRer10 transcript counting and gene cell barcode matrix generation. Before the downstream analysis cells with unique detected genes lower than <2 000 and higher than 6 000 genes were discarded and cells with high percentage of mitochondrial genes >5% were removed too. Subtypes of cells were identified with Seurat 3.0 through the integrating of control and mKD embryos at dome and shield stages with setting control embryos as reference. To make the data comparable samples we performed SCTransform normalization separately for each dataset. To further reduce the bais caused by sequencing depth of scRNA seq and percentage of mitochondrial genes UMI variance and percent.mt were regressed out from SCTransform normalized data matrix by linear model with negative binomial distribution. All ChIP seq reads were aligned to the zebrafish reference genome danRer7 using Bowtie2 version 2.2.2 Langmead and Salzberg 2012 with the parameters –t –q –N 1 –L 25. All unmapped reads non uniquely mapped reads and PCR duplicates were removed. For downstream analysis the read counts were normalized by computing the numbers of reads per kilobase of bin per million of reads sequenced RPKM. To visualize the STAR ChIP seq signal in the UCSC genome browser each read was extended by 250 bp and the coverage for each base was counted. Replicates of STAR ChIP seq were pooled for the downstream analysis. The ATAC seq data were mapped to the zebrafish reference genome danRer7 using Bowtie2 version 2.2.2 Langmead and Salzberg 2012 with the parameters –t –q –N 1 –L 25. All unmapped reads non uniquely mapped reads and PCR duplicates were removed. For downstream analysis the read counts were normalized by computing the numbers of reads per kilobase of bin per million of reads sequenced RPKM. To minimize the batch and cell type variation RPKM values across whole genome were further Z score normalized. To visualize the CUT&RUN signals in the UCSC genome browser each read was extended by 250 bp and the coverage for each base was counted. Genome build: danRer7 Supplementary files format and content: Matrix for scRNA seq data gene expression table for total RNA seq data Bigwig and narrowPeak for ChIP seq and ATAC seq data	larva	Embryos at 5 dpf were euthanized with tricaine and tail was dissected carefully by tweezers. post brief grinding tissues froze at 80℃ for later usage.	The embryos were dechorionated manually by tweezers and transferred into 750 µl Trizol Invitrogen Cat 15596018. About 10 fresh embryos were transferred into Trizol and vortexed until no visible particles. 150 µl chloroform Amresco Cat 0757 was added and mixed thoroughly. The mixture was then transferred into phasemaker tube Invitrogen Cat A33248 and spun at 14 000 rpm for 15 min. Next the top phase was taken out from the tube added 1 µl LPA Sigma Cat 56575 and mixed well using pipettes. Then RNA was precipitated by adding 750 µl isopropanol Sigma Cat 59304 at 20℃ overnight. At next day the tube was spun at 14 000 rpm for 30 min and supernatant was removed. The pellet was washed with freshly 70% ethanol re suspend in 20 µl RNase free water and stored at 80℃ for later usage. NEBNext rRNA Depletion Kit NEB Cat E6310S was used to deplete ribosomal RNA according to the manufacture’s instruction. Briefly rRNA were hybridized with probes and digested with RNase H then excess probes were digested with DNase I. post that NEBNext RNA sample purification beads were used to purify rRNA depleted RNA. Purified RNA was fragmented before cDNA synthesis at 95℃ for 8 min. Double stranded cDNA was synthesized with NEB Next first strand NEB Cat E7771S and second strand synthesis modules NEB Cat E7550S then purified with Ampure XP beads Beckman Cat A63882. Synthesized cDNA was subjected to library preparation with NEBNext Ultr II DNA Library Prep Kit NEB Cat E7645S. DNA was end repaired adenylated and ligated to TruSeq sequencing adaptors. DNA were amplified using KAPA HF HotStart ReadyMix KAPABiosystem Cat RR2602. The amplified DNA was size selected using Ampure XP beads for 200 500 bp DNA fragments. All libraries were sequenced by Illumina Hi Seq 1500 or 2500 or XTen platform according to manufacturer’s instruction.	The dnmt1 mutant TL strain was used in the experiments. The adult fish were fed with live adult brine shrimp in the morning and evening. All embryos were raised in Holtfreter’s solution at 28.5C and staged as described previously Kimmel et al. 1995.	strain:TL\|genotype:dnmt1 / \|developmental stage:5dpf\|tissue:larva tail\|treatment:untreated	GSM5351821	GSM5351821: 5dpf tail dnmt1 zKO total RNA seq rep2; Danio rerio; RNA Seq	GSM5351821		1	The embryos were dechorionated manually by tweezers and transferred into 750 µl Trizol Invitrogen Cat 15596018. About 10 fresh embryos were transferred into Trizol and vortexed until no visible particles. 150 µl chloroform Amresco Cat 0757 was added and mixed thoroughly. The mixture was then transferred into phasemaker tube Invitrogen Cat A33248 and spun at 14 000 rpm for 15 min. Next the top phase was taken out from the tube added 1 µl LPA Sigma Cat 56575 and mixed well using pipettes. Then RNA was precipitated by adding 750 µl isopropanol Sigma Cat 59304 at 20℃ overnight. At next day the tube was spun at 14 000 rpm for 30 min and supernatant was removed. The pellet was washed with freshly 70% ethanol re suspend in 20 µl RNase free water and stored at 80℃ for later usage. NEBNext rRNA Depletion Kit NEB Cat E6310S was used to deplete ribosomal RNA according to the manufacture's instruction. Briefly rRNA were hybridized with probes and digested with RNase H then excess probes were digested with DNase I. post that NEBNext RNA sample purification beads were used to purify rRNA depleted RNA. Purified RNA was fragmented before cDNA synthesis at 95℃ for 8 min. Double stranded cDNA was synthesized with NEB Next first strand NEB Cat E7771S and second strand synthesis modules NEB Cat E7550S then purified with Ampure XP beads Beckman Cat A63882. Synthesized cDNA was subjected to library preparation with NEBNext Ultr II DNA Library Prep Kit NEB Cat E7645S. DNA was end repaired adenylated and ligated to TruSeq sequencing adaptors. DNA were amplified using KAPA HF HotStart ReadyMix KAPABiosystem Cat RR2602. The amplified DNA was size selected using Ampure XP beads for 200 500 bp DNA fragments. All libraries were sequenced by Illumina Hi Seq 1500 or 2500 or XTen platform according to manufacturer's instruction.	GEO Accession:GSM5351821	RNA-Seq	TRANSCRIPTOMIC	cDNA	PAIRED	ILLUMINA	Illumina HiSeq 1500		SRP322195		loader:fastq load.py	5dpf_tail_dnmt1_zKO_total_RNA_seq_rep2_r1.fq.gz 5dpf_tail_dnmt1_zKO_total_RNA_seq_rep2_r2.fq.gz	fastq fastq	12390971100.0	41303237.0	GSM5351821 r1	0:150 1:150	A:3159992851;C:3017116837;G:3080231294;T:3133156041;N:474077	150	150			3159992851	3017116837	3080231294	3133156041	474077	SRX11041466	SRS9110394	SRA1239365	GEO	THU-PKU Center for Life Sciences, Tsinghua University	2	0.93558	0.94895	0.11597	0.11647	0.71041	0.71403	0.45173	0.45741	150	150	B	B	biological fallback assumption	illumina	hiseq_era	unknown	rrna_depletion	nebnext	sc_generic	single_cell_generic	generic-scrnaseq-only		China	2021-06-01	Larval	Larval	Tail	Multi-system