Chromatin Immunoprecipitation Assay Protocol

ChIP assay was performed using OneDay ChIP kit and Shearing ChIP kit (Diagenode, Denville, USA) according to the manufacturer’s protocol. After cross-linking, cells were fixed in formaldehyde and DNA was sheared to small fragment sizes. After incubation with specific antibodies against H3K9me3 (Abcam) and IgG as a negative control (Diagenode, Denville, USA), reversed cross-linking and proteinase K treatment were carried out. 5 μl of eluted DNA was added to the reaction mixture containing the primer pair (300 nmol/l each) (Supplemental Table 3), a ROX passive reference dye (Bio-Rad, Hercules, USA) and diluted 2× SYBR green Supermix (Bio-Rad, Hercules, USA) in a final volume of 25 μl for each PCR reaction. The real-time PCR reactions were performed in a 96-well reaction plate using the Mx3000P QPCR System (Stratagene, Santa Clara, USA). PCR reaction was stopped when the fluorescent signal increased over the threshold and electrophoresis of PCR products were done on a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, USA) according to the manufacturer’s protocol. Electrophoresis results are shown as virtual gel images as described in our previous publications72 (link)73 (link)74 (link).