GSM9396559: anti-P300-CCS1477; Homo sapiens; OTHER
Cells were harvested and washed twice with wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine and EDTA-free protease inhibitor cocktail). The cells were then incubated overnight with the rabbit anti-P300 antibody (1:50, Cell Signaling, Cat# 54062, RRID:AB_2799450) in the buffer (Wash buffer supplemented with 0.05% digitonin and 2 mM EDTA) at 4 °C. The following day, the supernatant was removed by centrifugation and the cell pellets were washed once with Dig-wash buffer (Wash buffer containing 0.05% digitonin). The 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 washes, the cell pellets were resuspended in 100 μl Dig-wash buffer with 2 μl of 100 mM CaCl2, incubated at 0 °C for 30 min and then the reactions were stopped by the addition of 100 μl 2×STOP buffer (340 mM NaCl, 20 mM EDTA pH 8.0, 4 mM EGTA, 0.05% digitonin, 50 μg/mL RNase A, 50 μg/mL glycogen and 2 pg/mL heterologous spike-in yeast DNA). The supernatant DNA was collected after centrifugation and further purified using phenol–chloroform–isoamyl alcohol (Sigma, Cat#: p3803), chloroform extraction and ethanol precipitation. The CUT&RUN-seq library construction was performed using 10 ng CUT&RUN DNA and a NEBNext ultra DNA library prep kit (NEB, Cat#: E7645); the CUT&RUN-seq libraries were sequenced in a 50 bp paired-end sequencing on a NextSeq 1000 system (Illumina), using Illumina's NextSeq 1000 P2 Reagents (#20046811) following the manufacturer's instructions.