GSM7734444: ChIP, Nanog, NANOGON, rep2; Mus musculus; ChIP-Seq
Briefly, cells were collected after trypsinization and fixed with 2 mM DSG in PBS for 50 min at room temperature on a rotating platform. Subsequently, cells were pelleted, and fixed for 10 min with 1 % formaldehyde on a rotating platform. The reaction was stopped using 200 mM Tris-HCl pH 8.0 for 10 min on a rotating platform. Tubes were filled with 1 % FBS in PBS and cells were spun down. Nuclei were extracted by resuspending the cell pellet twice in LB1 (50 mM HEPES-KOH pH 7.4, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10% Glycerol, 0.5% NP-40, 0.25% Triton X-100), incubated for 10 min at 4°C and with gentle shaking at 100 rpm, and pelleted. The nuclei were then resuspended in LB2 (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA), incubated for 10 min at 4°C and with gentle shaking at 100 rpm, and spun down. To wash nuclei, SDS shearing buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.15% SDS) was added twice without disturbing the pellet, the tube was rolled to rinse the walls and spun down. All buffers were supplemented with a 1:100 dilution of protease inhibitor cocktail. Chromatin was sheared for 20 min at 5% duty, 140 W, 200 cycles, or for samples with 50,000 cells for 10 min, 10% duty, 75 W, 200 cycles in a Covaris E220 focused ultrasonicator. To remove the insoluble material, chromatin was spun down and the supernatant was transferred to a new tube. As total input control and to evaluate the sonication efficiency, a few microliters of chromatin were incubated with 1X TE buffer pH 8.0 and 10 ng/µl RNAse A at 37°C for 30 min. Proteinase K was added at 400 ng/µl and the reaction was incubated for another 30 min at 55°