ChIP-seq of H3K27ac in K562 with rTetR-VP48 constitutively expressed and 4xTetO reporter under p300 inhibitor A485 conditions
ChIP-seq was performed as previously described (Marinov 2017) with modifications. For each ChIP reaction, 2x10^7 cells were used as input together with a spike-in of mouse chromatin used for orthogonal normalization. Cells were crosslinked with 1% formaldehyde for 15 min at room temperature, followed by quenching in glycine (final concentration 0.125 M). Cells were then centrifuged, resuspended in 1xPBS, centrifuged again, and stored at -80C. On the first day of the ChIP procedure, 10 uL Protein A Dynabeads (Thermofisher 10002D) were added to DNA Lo-Bind tubes and washed three times on a magnetic rack with 1 mL mg/mL BSA. Beads were resuspended in 1 mL BSA, 5 ug anti-H3K27ac antibody (Abcam ab4729) were added, and the antibodies were coupled to the beads overnight on a rotor at 4 C. On the second day, crosslinked chromatin was resuspended in 1 mL Farnham Lysis Buffer (FLB; 5 mM HEPES pH 8.0, 85 mM KCl, 0.5% NP-40/IGEPAL, Roche Protease Inhibitor Cocktail), centrifuged, then resuspended in 1 mL FLB and centrifuged again, then resuspended in 880 uL RIPA buffer (1x PBS, 1% NP-40, 0.5% Sodium deoxycholate, 0.1% SGS, Roche protease inhibitor cocktail), and sheared using Covaris E220 Focused-Ultrasonicator. Beads were washed again three times with 1 mL BSA on a magnetic rack, resuspended in 100 uL BSA, and the chromatin in RIPA buffer was added to them, then incubated overnight at 4 C on a rotor. On the third day, beads were washed five times with LiCl buffer (10mM Tris pH 7.5, 500 mM LiCl, 1% NP-40, 1% Sodium deoxycholate) for 10 minutes on a rotator at 4 C followed by removal of the buffer using a magnetic rack, then washed once with 1 mL 1x TE buffer, and resuspended in 200 uL IP elution buffer (1% SDS, 0.1 M NaHCO3). Chromatin was eluted off the beads by incubation at 65 C followed by centrifugation at max speed for 3 minutes, and transfer to fresh DNA Lo-Bind tubes. Crosslinks were reversed by addition of 2 uL Proteinase K (Promega) and incubation at 65 C for 12-16 hours in a Thermomixer. DNA was purified by adding an equal volume (200 uL) 25:24:1 Phenol:Chloroform:Isoamyl alcohol, vortexing, and centrifugation for 3 minutes at max speed. The top phase was then purified using the MinElute kit (Qiagen), eluting in 50 uL 55C EB buffer. Sequencing libraries were prepared using the NEBNext Ultra II kit (NEB E7645) following the manufacturers instructions. Sequencing was performed on a NextSeq in 2x38 bp format.