ChIP assay

ChIP assay was performed as previously described⁴¹. Briefly, MCF-7 cells were crosslinked in 1% formaldehyde solution for 10 min at room temperature, followed by the addition of 125 mM of glycine for 5 min. ChIP assay was performed with ChIP assay kit (26156, Thermo) according to the manufacturer’s instructions. The nucleoprotein complexes were digested to yield DNA fragments ranging from 200 to 500 bp using the Micrococcal Nuclease in the kit. Two micrograms of normal IgG were used as the negative control while anti-Bmi1 was used for each immunoprecipitation. The immunoprecipitates were eluted and reverse crosslinked, after which the DNA fragments were purified. Immunoprecipitated and input DNAs were subjected to qRT–PCR analysis. The primers used for amplifying the Bmi1 recognizing site were previously validated²⁸ and are as follows: forward - 5-CCCATTTTCCTATCTGC-3; reverse - 5-CTAGTTCAAAGGATTCC-3.