H2a z

HEK293T cells (1x10⁸) were crosslinked with 1% formaldehyde for 15 minutes and quenched with 0.125 M glycine. Cell lysis and chromatin preparation were performed as previously (Lee et al., 2006; Vo et al., 2017). For sonication, the sample was resuspended in 2 mL of ChIP buffer #3 and sonicated on a Covaris E220 sonicator for 5 minutes, 200 cycles per burst, 140W peak intensity pulse and 20% duty factor. Following sonication, samples were cleared by centrifugation at 20,000xg for 15 minutes. Chromatin was then diluted to a concentration of 0.4 mg/mL and Triton X-100 added to a final concentration of 1%. For each ChIP binding reaction, 0.4 mg of chromatin was incubated with 10 μL of antibody overnight at 4°C. The following day, 20 uL of Protein-A/G plus beads (Santa Cruz sc-2003) were added to each ChIP and incubated for 2 hours. Beads were washed 5 times in ChIP wash buffer and 2 times in TEN buffer. ChIP’ed DNA was purified using phenol: chloroform as previously (Lee et al., 2006; Vo et al., 2017). Full references available in Supplemental Information. Antibodies used: H2A.Z (Cell Signaling Technologies, CST, #2718S) Lot #2; H2A.Z (Abcam, #ab4174) Lot# GR3198864-2; H3.3 (EMD/Millipore, #09-838) Lot #3310680; H4K16ac (CST, #13534S) Lot #3; H3K79me2 (CST, #5427S) Lot#4. Western blot analysis was performed using 1:1000 dilution of these antibodies.