GSM3963204: ChIP_MD-OCT4_S_H3K27ac_2; Mus musculus; ChIP-Seq
Roughly 10 million cells per sample were collected after trypsinization and fixed with 2 mM disuccinimidyl glutarate (Thermo Fisher Scientific #20593) in PBS for 50 minutes at room temperature, spun down at 600g for 5 minutes and washed once with PBS. Cells were then treated with 1% formaldehyde (Axon Lab #A0877,0500) for 10 minutes at room temperature and quenched with 200mM Tris-HCl pH 8.0 for 10 minutes, washed with PBS and spun down. For ZHBTc4 YPet-MD SNAP-MD-OCT4 and SNAP-MD*-OCT4 cells, cells were subsequently resuspended in cold PBS with 1% FBS and at least 500'000 cells per cell cycle phase were sorted. Fixed cell pellets were kept on ice and resuspended in LB1 (50mM HEPES-KOH pH 7.4, 140mM NaCl, 1 mM EDTA, 0.5mM EGTA, 10% Glycerol, 0.5% NP-40, 0.25% Triton X-100), incubated 10 min at 4°C, spun down at 1700g, and resuspended in LB1 a second time, spun down and resuspended in LB2 (10mM Tris-HCl pH 8.0, 200mM NaCl, 1 mM EDTA, 0.5mM EGTA), incubated for 10 min at 4°C, spun down and washed without disturbing the pellet three times with SDS shearing buffer (10mM Tris-HCl pH 8.0, 1mM EDTA, 0.15% SDS) and finally resuspended in SDS shearing buffer. All buffers contained Protease Inhibtor Cocktail (Sigma #P8340-1ML) at 1:100 dilution. Chromatin was sonicated for 20 min at 5% duty cycle, 140 W, 200 cycles on a Covaris E220 focused ultrasonicator. Sonicated chromatin was equilibrated to 1% Triton X-100 and 150 mM NaCl and incubated with each antibody overnight at 4°C. Antibodies used were anti-BRG1 (Abcam #ab110641) at 5 μg per 10 million cells, anti-OCT4 (Cell Signaling Technology #5677S) at 20 μl per 10 million cells, and anti-H3K27ac (Abcam #ab4729) at 2 μg/25 μg chromatin. Protein G Dynabeads (Thermo Fisher Scientific #10003D) were blocked with 5 mg/ml BSA in PBS, added to chromatin, and incubated at 4°C for 3 hours. Beads were washed twice with Low Salt wash buffer (10mM Tris-HCl pH 8.0, 150mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.15% SDS, 1 mM PMSF), once with High Salt wash buffer (10mM Tris-HCl pH 8.0, 500mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.15% SDS, 1 mM PMSF), once with LiCl wash buffer (10mM Tris-HCl pH 8.0, 1mM EDTA, 0.5mM EGTA, 250mM LiCl, 1% NP40, 1% sodium deoxycholate, 1mM PMSF), and finally with 1X TE (10 mM Tris pH 8.0, 1 mM EDTA) before being resuspended in ChIP Elution buffer (10 mM Tris pH 8.0, 1 mM EDTA, 1% SDS, 150 mM NaCl) with 400 ng/μl Proteinase K (Qiagen #19131) and reverse-crosslinked overnight at 65°C. DNA was purified using a MinElute PCR purification kit (Qiagen #28004) Libraries were prepared with NEBNext Ultra II DNA Library Prep Kit (NEB #E7645S) kit with insert size selection of 250 bp