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| Status |
Public on May 13, 2021 |
| Title |
DNMT3A1-K299I_ChIP-seq_sgEzh2 |
| Sample type |
SRA |
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| Source name |
C3H10T1/2 cells
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| 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 K299I
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| Growth protocol |
C3H10T1/2 cells were cultured in Dulbecco's modified Eagles' medium (DMEM, Invitrogen) with 10% fetal bovine serum (FBS, Sigma).
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| 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
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| 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
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| Submission date |
Feb 01, 2021 |
| Last update date |
May 14, 2021 |
| Contact name |
Jacek Majewski |
| Organization name |
McGill University
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| Department |
Human Genetics
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| Street address |
740 Dr Penfield Ave
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| City |
Montreal |
| State/province |
Quebec |
| ZIP/Postal code |
H3A 0G1 |
| Country |
Canada |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE147879 |
Two competing mechanisms of DNMT3A recruitment regulate the dynamics of de novo DNA methylation at CpG islands |
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| Relations |
| BioSample |
SAMN17732599 |
| SRA |
SRX10000623 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5058909_DNMT3A1_K299I_200_sgEzh2.bw |
81.0 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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