title: GSM5058872: DNMT3A1-K299I_ChIP-seq_parental; Mus musculus; ChIP-Seq
library_construction_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