Chromatin Immunoprecipitation (ChIP)

ChIP was performed as previously described (31) using the ChIP-grade antibody mouse anti-H3K9me2 (Abcam). Cells were fixed in 1% paraformaldehyde for 5 min at room temperature and then quenched with 1 ml of 2.5 M glycine for 5 min at room temperature. After washing, cells were lysed in 1 ml ChIP lysis buffer (50 mM HEPES- KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, pH 8.0, 1% Triton X-100, and 0.1% deoxycholate with 0.1 mM PMSF and the EDTA-free Protease Inhibitor Cocktail). Samples were incubated on ice for 10 min and then centrifuged at 3,000 rpm for 3 min at 4°C. The pellet was re- suspended in 500 μl lysis buffer 2 (10 mM Tris, pH 8.0, 200 mM NaCl, 1 mM EDTA, and 0.5 mM EGTA with 0.1 mM PMSF and the EDTA-free Protease Inhibitor Cocktail) and incubated at room temperature for 10 min. Samples were centrifuged at 3,000 rpm for 5 min at 4°C. Next, the pellet was resuspended in 300 μl lysis buffer 3 (10 mM Tris, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% DOC, and 0.5% N-lauroylsarcosine with 0.1 mM PMSF and the EDTA-free Protease Inhibitor Cocktail). Cells were sonicated using a Branson Sonifier 250 for four cycles of 10 seconds on 50 seconds off. Next, 30 μl of 10% Triton X-100 was added to each tube, and then samples were centrifuged at max speed for 15 min at 4°C. 50 μl of the antibody bead conjugate solution was added to the supernatant, and chromatin was immunoprecipitated overnight on a rotator at 4°C. The following washes were performed: ChIP lysis buffer, ChIP lysis buffer + 0.65 M NaCl, wash buffer (10 mM Tris-HCl, pH 8.0, 250 mM LiCl, 0.5% NP-30, 0.5% deoxycholate, and 1 mM EDTA, pH 8.0), and TE (10 mM Tris-HCl, pH 8.0, and 1 mM EDTA, pH 8.0). DNA was eluted with TES (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, pH 8.0, and 1% SDS) for 30 min at 65°C. Reversal of cross-linking was performed by incubating samples overnight at 65°C. Proteins were digested using 1 mg/ml proteinase K and incubating at 37°C for 5 h. Finally, the DNA was purified using the Wizard SV Gel and PCR Clean Up kit (Promega). Immunoprecipitated DNA was analyzed by qPCR using iTaq Universal SYBR® Green Supermix (BioRad). Conditions for amplification were: 5 min at 95°C, 40 cycles of 95°C for 10 sec and 30 sec with 62°C annealing temperature. Enrichment of H3K9me2 was determined by normalizing to a gene desert region (31). Primer sets used for ChIP-qPCR are detailed in Table S1.