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Sample GSM2465454

Query DataSets for GSM2465454

Status

Public on Jan 01, 2018

Title

Dnmt3ab_WT_H3K4me3_rep3

Sample type

SRA

Source name

Germinal vesicle oocytes

Organism

Mus musculus

Characteristics

background strain: C57BL6/Babr; 129
time-point in reprogramming: postnatal day 25
cell number: ~250
cell type: Germinal vesicle oocytes
antibody amount: 250ng
genotype: Dnmt3a floxed/floxed, Dnmt3b floxed/floxed, Zp3 -ve

Growth protocol

Ovaries were collected from 5, 10, 15, and 25 day-old C57BL/6Babr or 21 or 25-day-old Mll2Gdf9cKO or 25-day-old Dnmt3a/bZp3cDKO mice and digested using 2mg/mL collagenase and 0.02% trypsin solution, gently agitating at 37°C for 20-30 minutes. Oocytes were hand collected and washed in M2 medium (Sigma), followed by 2 consecutive washes in PBS.

Extracted molecule

genomic DNA

Extraction protocol

The ChIP-seq libraries for all samples were prepared using an ultra-low input native ChIP protocol, as previously described with the several modifications. Samples were thawed on ice and permeabilised with 0.1% Triton-X/0.1% deoxycholate in PBS on ice. MNase digestion was completed using 200U of micrococcal nuclease in prepared digestion buffer, as per recommendations in the Brind’Amour protocol. Samples were digested at 21°C for 7.5 minutes and the reaction was stopped with EDTA. Chromatin samples were precleared with Protein A/G beads rotating at 4°C for 2 hours and antibodies were bound to beads rotating at 4°C for 3 hours. Chromatin was added to the antibody-bound beads and rotated overnight at 4°C. This was proceeded by two low-salt washes, one high-salt wash, and the DNA was then eluted from the beads at 65°C for 1.5 hours. DNA from immunoprecipitated chromatin or input was purified using spri purification using Sera-Mag carboxylate-modified Magnetic SpeedBeads at a 1.8:1 ratio. Library preparation was completed using the Diagenode MicroPlex Library Preparation kit v2 using Sanger 8-base indices for multiplexing, as per the manufacturers recommendations.
Cells for ChIP-seq were flash frozen in nuclear lysis buffer, cells for PBAT were flash frozen in PBS
The ChIP-seq libraries for all samples were prepared using an ultra-low input native ChIP protocol, as previously described with the several modifications. Samples were thawed on ice and permeabilised with 0.1% Triton-X/0.1% deoxycholate in PBS on ice. MNase digestion was completed using 200U of micrococcal nuclease in prepared digestion buffer, as per recommendations in the Brind’Amour protocol. Samples were digested at 21°C for 7.5 minutes and the reaction was stopped with EDTA. Chromatin samples were precleared with Protein A/G beads rotating at 4°C for 2 hours and antibodies were bound to beads rotating at 4°C for 3 hours. Chromatin was added to the antibody-bound beads and rotated overnight at 4°C. This was proceeded by two low-salt washes, one high-salt wash, and the DNA was then eluted from the beads at 65°C for 1.5 hours. DNA from immunoprecipitated chromatin or input was purified using spri purification using Sera-Mag carboxylate-modified Magnetic SpeedBeads at a 1.8:1 ratio. Library preparation was completed using the Diagenode MicroPlex Library Preparation kit v2 using Sanger 8-base indices for multiplexing, as per the manufacturers recommendations.
For single oocyte RNA profiling, four GV oocytes from Mll2 KO and WT mice were collected for each genotype. Cells were lysed, RNA reverse transcribed and amplified according to the protocol of SMARTer Ultra Low RNA Kit for Illumina Sequencing (Version 1, Clontech). Subsequently, samples were subjected to NEBNext Ultra DNA library preparation for Illumina using indexed adaptors (New England Biolabs). Resulting libraries were pooled in equimolar quantities for 75-bp single-read sequencing on Illumina HiSeq 2000, resulting in about 15-18 million reads per sample.
Post-bisulfite adaptor tagging (PBAT) was used to generate whole-genome bisulfite sequencing libraries using 100 collected oocytes. Briefly, cells are lysed with 0.5% SDS in EB buffer and bisulfite-treated with one-step modification procedure using the Sigma Imprint DNA Modification kit. The resulting DNA are purified using columns from Zymo EZ DNA Methylation Direct kit. First strand synthesis is then performed using Klenow Exo- with customized, streptavidin-conjugated adaptor containing standard Illumina adaptor sequences and 9 base pairs of random sequences (9N). This is followed by exonuclease I treatment and binding to Dynabeads M-280 Streptavidin beads. Samples are then subjected to second strand synthesis similar to the first strand synthesis, followed by 10 PCR amplification cycles using Phusion High-Fidelity DNA polymerase.
ChIP-bisulfite-seq was done on MNase-digested 10% input samples from the ChIP protocol, detailed in the corresponding ChIP-seq samples. Following DNA purification from chromatin using spri purification with Sera-Mag carboxylate-modified Magnetic SpeedBeads, samples were bisulfite converted using the Zymo EZ DNA Methylation Direct kit. First strand synthesis is then performed using Klenow Exo- with customized, streptavidin-conjugated adaptor containing standard Illumina adaptor sequences and 9 base pairs of random sequences (9N). This is followed by exonuclease I treatment and binding to Dynabeads M-280 Streptavidin beads. Samples are then subjected to second strand synthesis similar to the first strand synthesis, followed by 16-20 PCR amplification cycles using Phusion High-Fidelity DNA polymerase.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2000

Description

Dnmt3DKO.WT_H3K4me3_Me.Unme.domains_RPM.txt

Data processing

Raw FastQ sequence files were initially quality and adapter trimmed using Trim Galore v0.4.2 using default parameters (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/). Mapping of ChIP-Seq data was performed using Bowtie 2 v2.2.9 against the mouse GRCm38 genome assembly. The resulting hits were filtered to remove mappings with a MAPQ scores of < 20. Mapping of RNA-Seq data was performed using HISAT2 v2.0.5 against the mouse GRCm38 genome guided by known splice sites taken from Ensembl v68. Hits were again filtered to remove mappings with MAPQ scores of <20.
For PBAT data, reads were trimmed with Trim Galore using --clip_r1 9 and --clip_r2 9. Trimmed reads were first aligned to the mouse genome in paired-end mode to be able to count overlapping parts of the reads only once while writing out unmapped singleton reads; in a second step remaining singleton reads were aligned in single-end mode. Alignments were carried out with Bismark v0.16.3 (Krueger and Andrews, 2011) with the following set of parameters: a) paired-end mode: --pbat; b) single-end mode for Read 1: --pbat; c) single-end mode for Read 2: defaults. Reads were then deduplicated with deduplicate_bismark selecting a random alignment for position that were covered more than once. CpG methylation calls were extracted from the deduplicated mapping output, and methylation from single-end and paired-end mapping were merged into a single Bismark coverage report.
genome build: GRCm38
The Bismark CpG coverage report is tab-delimited, uses 1-based genomic coordinates for every covered cytosine position in the experiment and is in the following format: <chromosome> <start position> <end position> <methylation percentage> <count methylated> <count non-methylated>
processed data files format and content : "10_5_Domains_75_25_NoOverlap_NoGaps_NoInterm_NoSmall_NoGaps" file contains the GV methylated and unmethylated domains used for analyses in this study.
processed data files format and content : "RNAseq_groups_oocytemRNA_norep_RPKM_corrDNAcont" file contains RNA-seq quantitation for two groups: Mll2 WT oocytes and Mll2 cKO oocytes (each a replicate set of 4 single cell RNA-seq libraries). RPKM was quantitated in SeqMonk for oocyte transcriptome mRNA annotation (Veselovska et al. 2014) excluding repetitive elements, correcting for DNA contamination
processed data files format and content : "ESC_oocyte_technicaloptimisation_2kbrunning_RPM_nofilters" file contains ChIP-seq quantitation for individual replicates for H3K4me3 in 1000 ESCs and 500 ESCs used for validation of the ULI-nChIP-seq method and the antibody titration for 125ng,and 62.5ng of antibody for H3K27ac and 125ng, 250ng and 500ng of H3K27me3 in GV oocytes (25-day old mice). Reads per million was quantitated in SeqMonk for 2kb running window probes, with no filters applied.
processed data files format and content : "Dnmt3DKO.WT_H3K4me3_Me.Unme.domains_RPM" file contains ChIP-seq quantitation for H3K4me4 in GV oocytes (25-day old mice) from grouped Dnmt3a/b cDKO (N=4) and Dnmt3a/b WT controls (N=4). Reads per million was quantitated in SeqMonk for GV methylated and unmethylated domains.
processed data files format and content : "GO_and_Mll2_peaks" file contains peak and seedpeak calls for H3K4me3 in non-growing oocytes (NGO) (5-day old mice), growing oocytes (GO) (10-day old mice), germinal vesicle (GV) oocytes (15-day old mice) and GV oocytes (25-day old mice), Mll2 WT GV oocytes (25-day old mice), and Mll2 KO GV oocytes (25-day old mice) using Chromstar peak calling program. Temporal peak calling was done using a joint multivariate analysis (parameters: binsize = 1000bp, stepsize = 200bp, mode = differential). A cutoff of 1-10-4 was applied on the peak score (which is defined as the maximum posterior probability anywhere within a peak), to divide the resulting peak set into peaks with high H3K4me3 enrichment and seed-peaks with low H3K4me3 enrichment.
processed data files format and content : "bed_files_combinations_GV" file contains combinatorial peak for H3K4me3, H3K27ac, and H3K27me3 in germinal vesicle (GV) oocytes (25-day old mice) using Chromstar peak calling program. Combinatorial chromatin states were called using a combined multivariate analysis. A cutoff of 1-10-4 was applied on the posterior probability in each bin to yield a stringent set of peaks.
processed data files format and content : "Epiblast_combinations_and_peaks" file contains combinatorial peak for H3K4me3, H3K27ac, and H3K27me3 in E6.5 epiblast using Chromstar peak calling program. Combinatorial chromatin states were called using a combined multivariate analysis. A cutoff of 1-10-4 was applied on the posterior probability in each bin to yield a stringent set of peaks.
processed data files format and content : "bivalency_PGC_d05_d25_domains" file contains combinatorial peak for combined replicates for H3K4me3, H3K27me3 in non-growing oocytes (NGO) (5-day old mice), GV oocytes (25-day old mice), E11.5 primordial germ cells (Sachs et al. 2013). Combinatorial chromatin states were called using a joint multivariate analysis (parameters: binsize = 1000bp, stepsize = 200bp, mode=differential). A cutoff of 1-10-4 was applied on the peak score to select broad bivalent domains and subsequently only those domains were retained which contained clear enrichment, by applying a cutoff on the posterior probability of 1-10-4.
processed data files format and content : "OocyteH3K27ac_H3K27acGVpeaks_RPMcorrlength" file contains H3K27ac enrichment in grouped d5 NGOs and d25 GV oocytes for H3K27ac peaks called in GV oocytes using Chromstar. Enrichment was quantitated as reads per million, corrected for probe length.
processed data files format and content : "Oocyte_H3K27ac_5kbrunning_RPM_nofilters" file contains H3K27ac enrichment in grouped d5 NGOs and d25 GV oocytes for running 5kb windows. Enrichment was quantitated as reads per million.

Submission date

Jan 23, 2017

Last update date

May 15, 2019

Contact name

Felix Krueger

E-mail(s)

fkrueger@altoslabs.com

Organization name

Altos Labs

Department

Bioinformatics