ChIP Assay for Chromatin Immunoprecipitation

ChIP assay was performed as described previously with slight modifications [33 (link)]. Briefly, A549 cells were cross-linked with 1% formaldehyde for 10 min at room temperature. The cross-linking reaction was stopped by adding glycine stock solution to a final concentration of 0.125 M. DNA was sheared to an average size of ~500 bp using a Vibra cell sonicator (Sonics and Materials Inc., Newtown, CT, USA). The sonicated mixture was centrifuged at 14,000 rpm for 15 min at 4 °C, and the supernatant was collected. Immunoprecipitation was performed using 5 μg/sample of normal mouse IgG (Santa Cruz Biotechnology, Dallas, TX, USA), anti-Ago2 (Abcam, Cambridge, MA, USA), anti-H3K4me2, anti-H3K9me2, or H3ac (Cell Signaling Technology). The samples were incubated with Protein A/G Agarose suspension (Merck Millipore) for 2 h at 4 °C and sequentially washed with low salt, high salt, and TE buffer. Elutes were collected and reverse cross-linked at 65 °C overnight. Samples were subsequently treated with RNase A and Proteinase K followed by phenol/chloroform extraction and analyzed by PCR. PCR primers used for ChIP analysis are listed in Supplementary Table S1.