Chromatin immunoprecipitation (ChIP)

Cells (1.3 × 10⁷) were fixed in 1% formaldehyde for 6 min at room temperature. Glycine (125 mM) was added, and cells were harvested, washed in PBS, and kept at −80°C. For chromatin isolated pellet was suspended in 1 mL of buffer 1 (50 mM HEPES at pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% IGECAP CA-630, 0.25% Triton X-100, protease inhibitors). After rotating for 10 min at 4°C, nuclei were collected by spinning at 1700g for 5 min and then suspended in buffer 2 (10 mM Tris-HCl at pH 8, 200 mM NaCl, 5 mM EDTA, 2.5 mM EGTA, protease inhibitors). After another 10-min rotation at 4°C and spinning at 1700g for 5 min, nuclei were suspended in buffer 3 (10 mM Tris-HCl at pH 8, 5 mM EDTA, 2.5 mM EGTA, protease inhibitors). RNase A (20 mg/mL) and 0.5% lauroyl-sarcosine were added and rotated for 30 min at 37°C. Next, chromatin was sheared using Diagenode Bioruptor XL twice for 20 min each (with 30 sec on, 30 sec off cycles). Debris was separated by spinning at 15,000g for 15 min. Supernatant was diluted 1:2 with dilution buffer (30 mM Tris-HCl at pH 8, 300 mM NaCl, 2% Triton X-100). Input was removed, and 2 µg of antibodies was added to the lysate, respectively: negative control IgG, anti-G9a (R&D clone A8620A), anti-H3K9me2 (Abcam ab1220), or anti-H3K9Ac (Abcam ab4441). After overnight immunoprecipitation, magnetic protein G was added for 1 h, and then beads were washed five times with wash buffer (50 mM HEPES at pH 7.5, 10 mM EDTA, 0.5% sodium-deoxycholate, 1% IGEPAL CA-630, 0.5 M NaCl) and once with TEN (10 mM Tris-HCl at pH 8, 50 mM NaCl, 1 mM EDTA). Next, ChIP material was eluted by suspending beads in TES (50 mM Tris-HCl at pH 8, 1% SDS, 10 mM EDTA) and incubated for 20 min at 65°C with frequent vortexing. Cross-links were reversed overnight at 65°C while adding 40 µg of proteinase K. DNA was isolated by phenol extraction and ethanol precipitation and used for Q-PCR or library construction with New England Biolabs’ NebNext ChIP-seq library preparation master mix and following the manufacturer's instructions.