H 102

MDA-MB-231 cells were cultured in 150-mm dishes as previously described (Watanabe et al., 2014 (link)). ChIP was performed as previously described (Gu et al., 2013 (link)) with modifications. Briefly, cells were cross-linked with 1% formaldehyde for 10 minutes at room temperature, followed by quenching with 0.125 M glycine for 5 minutes at room temperature. After washing, chromatin was sheared to produce ~100–500-bp fragments using Bioruptor Sonicator (Diagenode Inc.) at “high” setting with 30-second ON and 30-second OFF cycles for a total of 30 minutes. A small aliquot of the recovered supernatant underwent subsequent reverse crosslinking and DNA purification, and the resulting DNA was used to assess concentration and shearing efficiency (input sample). Twenty-five μg of the crosslinked chromatin in the remaining supernatant was immunoprecipitated by overnight incubation at 4°C with control IgG (Santa Cruz Biotechnology, sc-2027) or anti-ZEB1 (Novus Biologicals, NBPI-88845), anti-ZEB1 (Santa Cruz Biotechnology, h-102), or anti-YAP1 (Cell Signaling Technology, 8418) antibodies. Antibody-chromatin immunocomplexes were then purified as previously described (Gu et al., 2013 (link)). ChIP DNA was recovered and purified using phenol: chloroform: isoamyl alcohol purification and then used for real-time PCR using primers listed in Table S6.