Sc 12984

With the following modifications, ChIP-exo was performed as previously described [24 (link)] with chromatin extracted from 50 million cells, ProteinG MagSepharose resin (GE Healthcare), and 5 μg of antibody directed against the H3K4me1, H3K4me3, H3K27ac, or Tal1 (Abcam ab8895, ab8580, ab4729, and Santa Cruz Biotech sc-12984, respectively). First, formaldehyde cross-linked cells were lysed with buffer 1 (50 mmol/L HEPES–KOH, pH 7.5; 140 mmol/L NaCl; 1 mmol/L EDTA; 10% glycerol; 0.5% Nonidet P-40; 0.25% Triton X-100) and washed once with buffer 2 (10 mmol/L Tris–HCL, pH 8; 200 mmol/L NaCl; 1 mmol/L EDTA; 0.5 mmol/L EGTA), and the nuclei were lysed with buffer 3 (10 mmol/L Tris–HCl, pH 8; 100 mmol/L NaCl; 1 mmol/L EDTA; 0.5 mmol/L EGTA; 0.1% sodium deoxycholate; 0.5% N-lauroylsarcosine). All cell lysis buffers were supplemented with fresh EDTA-free complete protease inhibitor cocktail (CPI, Roche, No. 11836153001). Purified chromatin was sonicated with a Bioruptor (Diagenode) to obtain fragments ranging from 100 bp to 500 bp. Triton X-100 was added to extract at 1% to neutralize sarcosine. Insoluble chromatin debris was removed by centrifugation, and sonication extracts were stored at −80°C until used for ChIP analysis.