ChIP and RNA immunoprecipitation

For ChIP, one 100-mm dish of cells was cross-linked for 10 min in 0.5% formaldehyde, and cross-links were quenched in 125 mM glycine for 5 min. Cells were collected (500g for 5 min) and resuspended in 400 µL of RIPA buffer (150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl at pH 8, 5 mM EDTA at pH 8). Samples were sonicated in a Bioruptor sonicator (30 sec on and 30 sec off) 10 times on high setting. Tubes were spun at 13,000 rpm for 10 min. Supernatant was then split into two and added to 30 µL of Dynabeads (Life Technologies) that had been incubated for 2 h with 3 µg of antibody or, as a control, mock-treated. Ten percent of the supernatant was kept for input. Immunoprecipitation was for 2–14 h at 4°C, and beads were then washed twice in RIPA, three times in high-salt wash buffer (500 mM NaCl, 1% NP40, 1% sodium deoxycholate, 100 mM Tris-HCl at pH 8.5), and once in RIPA. Samples were eluted (0.1 M NaHCO₃ + 1% SDS), and cross-links were reversed overnight at 65°C. DNA was purified by phenol chloroform extraction and ethanol precipitation. Samples were generally resuspended in 100 µL of water, with 1 µL used per PCR reaction. For RNA immunoprecipitation, cross-links were reversed for 45 min at 65°C. RNA was purified by phenol chloroform extraction and ethanol precipitation followed by DNase treatment and reverse transcription.