GSM5834066: H3K4me3; Mus musculus; OTHER
Cells were harvested by trypsinization and gently washed twice using wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, and EDTA-free Protease Inhibitor Cocktail). Cells were then incubated with the antibody at 4 °C overnight in antibody buffer (wash buffer supplemented with 0.05% digitonin and 2 mM EDTA). The next day, supernatant was removed by centrifugation and cell pellets were washed once with Dig-wash buffer (Wash buffer containing 0.05% digitonin). Cell pellets were then incubated with Protein A MNase (700 ng/ml in Dig-wash buffer) for 1 h by rotation at 4 °C. After three times of washes, cell pellets were resuspended in 100 ul Dig-wash buffer with 2 ul 100 mM CaCl2 and incubated at 0 °C for 30 min; reactions were stopped by addition of 100 ul 2× STOP buffer (340 mM NaCl, 20 mM EDTA pH 8.0, 4 mM EGTA, 0.05% digitonin, 50 ug/ml RNase A, 50 ug/ml glycogen). The supernatant DNA was collected after centrifugation and further purified using phenol–chloroform–isoamyl alcohol (Sigma, Cat# p3803) extraction and ethanol precipitation. Purified DNA was used for library construction using the NEBNext Ultra DNA Library Prep Kit (NEB, Cat# E7645) following the manufacturer's instructions.