GSM5990277: TKO_Parent_H3K36me2_ChIP; Mus musculus; ChIP-Seq
MEF-depleted TKO ESCs were crosslinked with 1% formaldehyde (Sigma) for 10 min at room temperature with rotation and then quenched with 125 mM glycine. Cells were pelleted, washed twice with cold PBS, snap frozen on dry ice, and stored at −80 °C until use. To perform ChIP, cells were first resuspended in ChIP lysis buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1% sodium dodecyl sulfate (SDS), 0.25% sodium deoxycholate (NaDOC), 1× Halt protease inhibitor) and incubated on ice for 10 min. Then, ChIP dilution buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.01% SDS, 0.25% Triton X-100) was added and the lysate was sonicated using a Branson sonifier (0.7 s on, 1.3 s off, 5 min total on, 50% amplitude), followed by centrifugation to clarify lysates. The supernatant was further diluted with ChIP dilution buffer and a sample was reserved for ChIP input. The diluted lysate was aliquoted and incubated with the appropriate antibodies overnight at 4 °C with rotation. Protein G dynabeads were then added and the samples were incubated for 2.5 h at 4 °C with rotation. The beads were isolated and washed twice with ChIP wash buffer A (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 0.1% NaDOC, 1% Triton X-100), once with ChIP wash buffer B (10 mM Tris-HCl, pH 8.0, 250 mM LiCl, 0.5% NaDOC, 0.5% Triton X-100), and once with ChIP wash buffer C (10 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1 mM EDTA). DNA was eluted by incubation (65 °C, 1 h, 1000 rpm shaking) with ChIP elution buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.1% SDS). Each sample was then treated with RNase and Proteinase K and purified by 2× SPRI. 2.5 ng each sample was subjected to end-repair (End-It DNA End-Repair kit, Lucigen), A-tailing (Klenow fragment, New England Biolabs), ligation to barcoded adapters (KAPA), and PCR library amplification (KAPA library amplification primers; NEB Ultra 2× master mix), followed by double-sided SPRI size selection.