{"database": "metadata", "table": "run_metadata", "rows": [[64443, "SRR14703446", "SRX11041467", "SRS9110397", "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 \u201cdememorization\u201d 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.", null, "pubmed:34936444", null, "Dome ctrl scRNA seq rep1", "GSM5351822", null, "source name:embryo|strain:AB|genotype:WT|developmental stage:Dome|tissue:embryo|treatment:microinjection with Rhodamine B and control MO at oogenesis III stage GV", "Dome ctrl scRNA seq rep1", "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 \u2013t \u2013q \u2013N 1 \u2013L 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 \u2013t \u2013q \u2013N 1 \u2013L 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 \u00b5g/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 \u00b5g/ml tricaine  10 unit/ml penicillin and 10 \u00b5g/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 standard control MO cMO were 5 ng per oocyte in the same assay. MOs were dissolved in RNase free water  and heated to 65\u00b0C for 10 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 \u00b5l DMEM/F12 media. Dissections were performed for up 15 min. The volume of DMEM/F12 meida containing embryos was adjusted to 200 \u00b5l  then cells were mechanically dissociated by flicking the tube 30 times and pipetting mixture 10 times through a 200 \u00b5l 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 \u00b5l of PBS containing 0.1% BSA and 20% Optiprep StemCell  Cat 07820  aiming for a concentration above 100 cells/\u00b5l. Cells were then passed through a cell sieve 100 \u00b5m for dome stage and 70 \u00b5m 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\u2019s 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\u2019s 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 control MO at oogenesis III stage GV", "GSM5351822", "GSM5351822: Dome ctrl scRNA seq rep1; Danio rerio; RNA Seq", "GSM5351822", null, "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 \u00b5l DMEM/F12 media. Dissections were performed for up 15 min. The volume of DMEM/F12 meida containing embryos was adjusted to 200 \u00b5l  then cells were mechanically dissociated by flicking the tube 30 times and pipetting mixture 10 times through a 200 \u00b5l 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 \u00b5l of PBS containing 0.1% BSA and 20% Optiprep StemCell  Cat 07820  aiming for a concentration above 100 cells/\u00b5l. Cells were then passed through a cell sieve 100 \u00b5m for dome stage and 70 \u00b5m 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:GSM5351822", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "HiSeq X Ten", null, "SRP322195", null, "loader:fastq load.py", "Dome_ctrl_scRNA_seq_rep1_1_r1.fq.gz Dome_ctrl_scRNA_seq_rep1_1_r2.fq.gz", "fastq fastq", 9940355100.0, 33134517.0, "GSM5351822 r1", "0:150 1:150", "A:2457125197;C:1850376601;G:1936367439;T:3696137056;N:348807", 150, 150, null, null, 2457125197, 1850376601, 1936367439, 3696137056, 348807, "SRX11041467", "SRS9110397", "SRA1239365", "GEO", "THU-PKU Center for Life Sciences, Tsinghua University", 2, 0.34261, 0.88683, 0.08556, 0.08811, 0.9516, 0.79054, 0.59718, 0.54828, 150, 150, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "China", "2021-06-01", "Blastula", "Embryo", "Embryo Imprecise", "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": ["64443"], "units": {}, "query_ms": 7.664621996809728}