ChIP Assay Protocol with Antibody Immunoprecipitation

ChIP assays were performed with a ChIP assay kit (Millipore, NY) by following the protocol provided by the manufacturer with slight modifications as previously described [51 (link)]. Briefly, 5 million cells were fixed with 1% formaldehyde and then sonicated for 180 s (10 s on and 10 s off) on ice using a Branson sonicator with a 2-mm microtip at 40% output control and 90% duty cycle settings. The sonicated chromatin was immunoprecipitated with specific antibodies to CTCF, SUZ12, and dimethyl-H3-K27 (lysine 27 of histone H3)(Cell Signaling, MA). Anti-IgG was used as the ChIP control in parallel with testing samples. ChIP DNAs were quantitated by qPCR using target gene primers (Supplementary Table 2). For comparison, the ChIP data are presented as relative values by normalizing to PCR signals of input DNA (i.e. ratio of the ChIP over the input).