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Sample GSM5058915 Query DataSets for GSM5058915
Status Public on May 13, 2021
Title DNMT3A1-D333N_ChIP-seq_sgEzh2
Sample type SRA
 
Source name C3H10T1/2 cells
Organism Mus musculus
Characteristics chip antibody: HA (ThermoFisher, #88836)
cell line: C3H10T1/2
cell type: C3H embryo-derived mesenchymal progenitor cells
genotype: sgEzh2 line expressing FLAG-HA tagged DNMT3A1 D333N
Growth protocol C3H10T1/2 cells were cultured in Dulbecco's modified Eagles' medium (DMEM, Invitrogen) with 10% fetal bovine serum (FBS, Sigma).
Extracted molecule genomic DNA
Extraction protocol Cross-linking ChIP in MSCs was performed as described previously (Weinberg et al. 2019) using ~2x107 cells per immunoprecipitation. Prior to fixation, media was aspirated and cells washed once with PBS. Cells were cross-linked directly on the plate using 1% paraformaldehyde for 5 min at room temperature with gentle shaking. Glycine was added to quench (final concentration 125 mM, incubated for 5 min at room temperature), then cells were washed once with cold PBS, scraped off the plates, and pelleted. To obtain a soluble chromatin extract, cells were resuspended in 1 mL LB1 (50 mM HEPES, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, 1x Complete protease inhibitor) and incubated rotating at 4°C for 10 min. Samples were centrifuged, resuspended in 1 mL LB2 (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1x Compete protease inhibitor), and incubated rotating at 4°C for 10 min. Finally, samples were centrifuged, resuspended in 1 mL LB3 (10 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na deoxycholate, 0.5% N-lauroylsarcosine, 1% Triton X-100, 1x Complete protease inhibitor), and homogenized by passing two times through a 27-gauge needle. Chromatin extracts were sonicated for 8 min (anti-HA ChIP) or 12 min (anti-histone PTM ChIP) using a Covaris E220 focused ultra-sonicator at peak power 140, duty factor 5, and cycles/burst 200. The lysates were incubated with 100 μl Pierce anti-HA beads (Themo Scientific, 88836) or 75 μl protein A Dynabeads (Invitrogen) bound to anti-H2AK119Ub (Cell Signaling, 8240) or anti-H3K27me3 (Cell Signaling Tech, 9733) antibodies and incubated overnight at 4°C with 5% kept as input DNA. Magnetic beads were sequentially washed with low salt buffer (150 mM NaCl; 0.1% SDS; 1% Triton X-100; 1 mM EDTA; 50 mM Tris-HCl), high salt buffer (500 mM NaCl; 0.1% SDS; 1% Triton X-100; 1 mM EDTA; 50 mM Tris-HCl), LiCl buffer (150 mM LiCl; 0.5% Na deoxycholate; 0.1% SDS; 1% Nonidet P-40; 1 mM EDTA; 50 mM Tris-HCl) and TE buffer (1 mM EDTA; 10 mM Tris-HCl). Beads were resuspended in elution buffer (1% SDS, 50 mM Tris-HCl pH 8.0, 10mM EDTA, 200 mM NaCl) and incubated for 30 min at 65°C. After centrifugation the eluate was reverse cross-linked overnight at 65°C. The eluate was then treated with RNaseA for 1 hr at 37°C and with Proteinase K (Roche) for 1 hr at 55°C and DNA was recovered using Qiagen PCR purification kit.
KAPA Hyper Prep kit
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina NovaSeq 6000
 
Data processing For ChIP-seq DNA input: Following alignment with BWA, bigwigs were generated with read coverage for 200-bp bins smoothed across 1-kb, computed with deepTools bamCoverage. Excludes mm10 ENCODE blacklist regions.
For ChIP-seq signal: Following alignment with BWA, bigwigs were generated with enrichment scores represented as input-normalized (log2-ratio), read-depth normalized signal per 200-bp bins, smoothed across 1-kb bins using deepTools. Excludes mm10 ENCODE blacklist regions. See publication Methods for details.
For ChIP-seq signal peaks: Following alignment with BWA, SICER2 was used to call broad H2AK119Ub peaks. Processed output is a BED file in the format "chrom, start, end, read-count".
For RRBS: Following alignment with Bismark, bedGraph files were generated representing methylation scores at CpGs of at least 10X read coverage, computed using MethylDackel. See publication Methods for details.
Genome_build: mm10 for Mus musculus, dm6 for Drosophila melanogaster
Supplementary_files_format_and_content: Filetype suffix explanation: bw = bigWig
 
Submission date Feb 01, 2021
Last update date May 14, 2021
Contact name Jacek Majewski
Organization name McGill University
Department Human Genetics
Street address 740 Dr Penfield Ave
City Montreal
State/province Quebec
ZIP/Postal code H3A 0G1
Country Canada
 
Platform ID GPL24247
Series (1)
GSE147879 Two competing mechanisms of DNMT3A recruitment regulate the dynamics of de novo DNA methylation at CpG islands
Relations
BioSample SAMN17732593
SRA SRX10000635

Supplementary file Size Download File type/resource
GSM5058915_DNMT3A1_D333N_200_sgEzh2.bw 82.6 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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