GSM6531705: batch1_H3K27me3_WT_rep2; Mus musculus; OTHER
105 cells were collected in NE buffer (20 mM HEPES pH 7.5, 0.5 mM spermidine, 10 mM KCl, 0.1% TritonX-100, 10% Glycerol, and 1 mM PMSF) and kept on ice for 10 min. 10 µl ConA beads which were pre-washed by binding buffer (20 mM HEPES pH 7.5, 10 mM KCl, 1 mM CaCl2, and 1 mM MnCl2) were added to each sample and incubated at room temperature for 10 min. After being washed with washing buffer (20 mM HEPES pH 7.5, 0.5 mM spermidine, 150 mM NaCl, and 0.1% BSA) once, beads were incubated with blocking buffer (20 mM HEPES pH 7.5, 0.5 mM spermidine, 150 mM NaCl, 0.1% BSA, and 2 mM EDTA) at room temperature for 5 min. Samples were incubated with diluted primary antibodies (1:100 dilution in washing buffer) at room temperature for 2 h. After washing with washing buffer once, secondary antibodies were added at a 1:100 dilution and incubated at room temperature for 30 min. PA-Tn5 transposons were then added and incubated at room temperature for 30 min. Beads were washed with washing buffer twice and incubated with 10 mM MgCl2 in washing buffer at 37 °C for 1h. The 30 μl tagmentation reaction was stopped by adding 2.25 μl 0.5 M EDTA, 2.75 μl 10% SDS, and 0.5 μl 20 mg/ml Proteinase K and incubated at 55 °C for 30 min, and then 70 °C for 20 min. DNA was purified by 0.9X of VAHTS DNA clean beads (VAHTS, Cat. #N411-03).