Ab4729

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.

ChIP coupled with massively parallel DNA sequencing (ChIP-seq) was performed as previously described^{31 (link),32 (link)}. The following antibodies were used for ChIP: anti-H3K27ac (Abam, ab4729) and anti GATA3(Santa Cruz, sc-22206X). For each ChIP, 10 μg of antibody was added to 3 ml of sonicated nuclear extract. Illumina sequencing, library construction and ChIP-seq analysis methods were previously described^{32 (link)}. ChIP-seq datasets are available under super series GSE65664, and relevant accession numbers are shown in Supplemental Table 2.

ChIP-seq or ChIP-qPCR was performed as described (78 (link)) using antibodies for H3K27ac (Abcam, ab4729), PPARG (Santa Cruz, sc-271392) or RXRA (Santa Cruz, sc-515928) in HL60, K562, MKPL-1, MV4–11, THP1 and U937 cells, respectively. ChIP-seq libraries were generated using NEBNext Ultra II DNA library prep kit following the manufacturer’s protocol (NEB), and sequenced on an Illumina NextSeq500 system using the 75bp high output sequencing kit. ChIP-seq raw reads were aligned to the human hg19 genome assembly using Bowtie2 (72 (link)) with the default parameters. Only tags that uniquely mapped to the genome were used.