run_metadata: 64434

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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
64434	SRR14703461	SRX11041470	SRS9110398	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		Dome dnmt1 mKD scRNA seq rep2	GSM5351825		source name:embryo\|strain:AB\|genotype:WT\|developmental stage:Dome\|tissue:embryo\|treatment:microinjection with Rhodamine B and dnmt1 MO at oogenesis III stage GV	Dome dnmt1 mKD scRNA 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	embryo	Briefly on the first day of OMIS Wu et al. 2018b adult females at 5 mpf 12 mpf were anesthetized in 550 µg/ml tricaine Sigma Cat A5040 in a petri dish. Then the fish was placed on a damp sponge with specific buffer 5.4 mM KCl 136.8 mM NaCl 4.2 mM NaHCO3 0.44 mM KH2PO4 0.25 mM Na2HPO4 and 0.5% wt/vol BSA. A cut was made on one side of belly to expose the ovary. The diluted MOs were microinjected into each oocyte. Rhodamine B Sigma Cat R8881 was co injected with MOs as a dye. post injection the wound on the belly was sewed with a surgical sewing needle carefully and quickly. Once the operation was done the female was transferred into fish water supplemented with 32 µg/ml tricaine 10 unit/ml penicillin and 10 µg/ml streptomycin HyClone Cat SV30010. Then the fish was transferred to fish water containing gradually reduced concentrations of tricaine. In the evening of the second day the female was paired with a wild type male. In the morning of the third day the pair started to chase and lay fertilized eggs naturally. The injected oocyte derived embryos were identified by co injected dye rhodamine B at very early developmental stages. The injection doses of dnmt1 MO were 5 ng per oocyte in the same assay. MOs were dissolved in RNase free water and heated to 65°C for 11 min before microinjection.	Cell dissociation protocol was based on a previously described method Farrell et al. 2018 with modifications to adapt it for 10x Genomics platform. Briefly dnmt1 mKD and control embryos were collected 20 min post fertilization and cultured as mentioned before. Then 15 embryos at dome or shield stages for each sample were transferred into plastic Petri dishes that had previously been coated with 2% agarose and soaked with DMEM/F12 media Gibco/Life Technologies Cat 11330032 at least 2hrs. Next embryos at desired stages were dechorionated and deyolked manually by forceps and transferred to a 1.5 ml Eppendorf tube with 50 µl DMEM/F12 media. Dissections were performed for up 15 min. The volume of DMEM/F12 meida containing embryos was adjusted to 200 µl then cells were mechanically dissociated by flicking the tube 30 times and pipetting mixture 10 times through a 200 µl tip. The volume was adjusted to 1 ml with PBS containing 1.0% BSA and spun to pellet cells at 300 g for 30s. The supernatant was removed and cells were resuspended in 80 µl of PBS containing 0.1% BSA and 20% Optiprep StemCell Cat 07820 aiming for a concentration above 100 cells/µl. Cells were then passed through a cell sieve 100 µm for dome stage and 70 µm for shield stage. Single cell RNA seq library was performed with Chromium Next GEM Single Cell kit 10x Genomics Cat PN 1000121 based on standard protocol. All libraries were sequenced by Illumina Hi Seq 1500 or 2500 or XTen platform according to manufacturer’s instruction.	The wild type AB strain was used in the experiments. The females post OMIS were fed with live adult brine shrimp thrice a day. All embryos were raised in Holtfreter’s solution at 28.5C and staged as described previously Kimmel et al. 1995.	strain:AB\|genotype:WT\|developmental stage:Dome\|tissue:embryo\|treatment:microinjection with Rhodamine B and dnmt1 MO at oogenesis III stage GV	GSM5351825	GSM5351825: Dome dnmt1 mKD scRNA seq rep2; Danio rerio; RNA Seq	GSM5351825		1	Cell dissociation protocol was based on a previously described method Farrell et al. 2018 with modifications to adapt it for 10x Genomics platform. Briefly dnmt1 mKD and control embryos were collected 20 min post fertilization and cultured as mentioned before. Then 15 embryos at dome or shield stages for each sample were transferred into plastic Petri dishes that had previously been coated with 2% agarose and soaked with DMEM/F12 media Gibco/Life Technologies Cat 11330032 at least 2hrs. Next embryos at desired stages were dechorionated and deyolked manually by forceps and transferred to a 1.5 ml Eppendorf tube with 50 µl DMEM/F12 media. Dissections were performed for up 15 min. The volume of DMEM/F12 meida containing embryos was adjusted to 200 µl then cells were mechanically dissociated by flicking the tube 30 times and pipetting mixture 10 times through a 200 µl tip. The volume was adjusted to 1 ml with PBS containing 1.0% BSA and spun to pellet cells at 300 g for 30s. The supernatant was removed and cells were resuspended in 80 µl of PBS containing 0.1% BSA and 20% Optiprep StemCell Cat 07820 aiming for a concentration above 100 cells/µl. Cells were then passed through a cell sieve 100 µm for dome stage and 70 µm for shield stage. Single cell RNA seq library was performed with Chromium Next GEM Single Cell kit 10x Genomics Cat PN 1000121 based on standard protocol. All libraries were sequenced by Illumina Hi Seq 1500 or 2500 or XTen platform according to manufacturer's instruction.	GEO Accession:GSM5351825	RNA-Seq	TRANSCRIPTOMIC	cDNA	PAIRED	ILLUMINA	Illumina HiSeq 1500		SRP322195		loader:fastq load.py	Dome_dnmt1_mKD_scRNA_seq_rep2_4_r1.fq.gz Dome_dnmt1_mKD_scRNA_seq_rep2_4_r2.fq.gz	fastq fastq	4380423600.0	14601412.0	GSM5351825 r4	0:150 1:150	A:1101017284;C:795734654;G:826725666;T:1656795538;N:150458	150	150			1101017284	795734654	826725666	1656795538	150458	SRX11041470	SRS9110398	SRA1239365	GEO	THU-PKU Center for Life Sciences, Tsinghua University	2	0.3746	0.87751	0.09966	0.13892	0.95668	0.78926	0.57333	0.52678	150	150	B	B	mate1-mate2 similar by mapping diff	illumina	hiseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x		China	2021-06-01	Blastula	Embryo	Embryo Imprecise	All anatomical structures