ChIP Assay for Human CML BC Cells

ChIP was performed on human CML BC cells harvested 24–48 h after treatment as previously described (Dawson et al., 2011 (link)). In brief, 10⁷ cells were fixed in 1% formaldehyde for 15 min at room temperature. The cross-linking reaction was stopped by addition of glycine (0.125 M final concentration) and additional incubation of 10 min. Cells were washed in PBS and cell pellets were lysed in lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.0, and 1 mM sodium orthovanadate and protease inhibitors). Chromatin was sonicated in a Bioruptor (Diagenode) sonicator and precleared for 1 h before immunoprecipitation in equal volumes of protein A and G beads (Dynabeads; Life Technologies). Immunoprecipitation was performed at 4°C overnight in modified RIPA buffer (1% Triton X-100, 0.1% deoxycholate, 0.1% SDS, 90 mM NaCl, 10 mM Tris-HCl pH 8.0, 1 mM sodium orthovanadate, and EDTA-free protease inhibitors) in the presence 2.5 µg of IgG (I5006; Sigma-Aldrich) or c-Myc (sc-764; Santa Cruz Biotechnology) antibodies and A/G beads. DNA was subsequently RNase treated, reverse cross-linked, and purified using QIAquick PCR purification kit (QIAGEN). ChIP-PCR was performed with SYBR Green PCR mastermix using the ABI Prism 7000 system (Applied Biosystems). The primers used can be found in Table S6.