KO_H3K27ac
HiChIP was performed as described in (Mumbach et al., 2016) with some modifications. Ten million cells were cross-linked for 10 min at room temperature with 1% formaldehyde in growth media and quenched in 0.125 M glycine. Cross-linked cell pellets were resuspended in 500 ul of ice-cold Hi-C lysis buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, and 0.2% IGEPAL CA-630) with protease inhibitor (Roche, #1169749800), and incubated at 4C for 30 min. Nuclei were pelleted and washed once with 500ul of ice-cold Hi-C lysis buffer. After removing supernatant, nuclei were incubated in 100ul of 0.5% SDS at 62C for 10 min. SDS was quenched by adding 285ul water and 50ul 10% Triton X-100, and nuclei were incubated for 15 min at 37C. After addition of 50ul of 10NEB Buffer 2 (Witt et al., B7002) and 375 U of MboI, chromatin was digested at 37 overnight. MboI enzyme was then inactivated by incubating at 62C for 20 min. To fill in the restriction fragment overhangs and mark the DNA ends with biotin, 52ul of fill-in master mix containing 37.5ul of 0.4 mM biotin-dATP (Invitrogen, #19524016), 1.5ul of 10 mM dCTP (Invitrogen, #18253013), 1.5ul of 10 mM dGTP (Invitrogen, #18254011), 1.5ul of 10 mM dTTP (Invitrogen, #18255018), and 10ul of 5 U/ul DNA Polymerase I, Large (Klenow) Fragment (Witt et al., M0210) was added and incubated at 37C for 1h with rotation. Proximity ligation was performed by incubating in a solution of 947ul of ligation master mix containing 150ul of 10X NEB T4 DNA ligase buffer (Witt et al., B0202), 125ul of 10% Triton X-100, 7.5ul of 20 mg/mL BSA (Sigma, #A7906), 10ul of 400 U/ul T4 DNA ligase (Witt et al., M0202) and 655.5ul of water at room temperature for 4h with rotation. After proximity ligation, nuclei were pelleted and resuspended in 1ml of ChIP sonication buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA pH 8.0, 1 mM EGTA pH 8.0, 1% Triton X-100, 0.1% sodium deoxycholate, and 0.1% SDS) with protease inhibitor. Nuclei were sonicated using a Covaris S220. 60ul of protein G magnetic beads were washed three times with sonication buffer, resuspended in 50ul of sonication buffer, and added to the sonicated chromatin and incubated for 1h at 4C with rotation. Beads were then separated on a magnetic stand and the supernatant was mixed with 7.5 ug of OCT4 or H3K27ac antibody, and incubated overnight at 4C with rotation. 60ul of protein G magnetic beads were washed and resuspended in 100ul of sonication buffer. Samples were incubated with the beads for 2h at 4C with rotation. Beads were then separated on a magnetic stand and washed three times with 1ml of high salt sonication buffer (50 mM HEPES-KOH pH 7.5, 500 mM NaCl, 1 mM EDTA pH 8.0, 1 mM EGTA pH 8.0, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS) followed by three times with 1ml of LiCl wash buffer (20 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 250 mM LiCl, 0.5% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS) and once with 1ml of TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 50 mM NaCl). Beads were then resuspended in 200ul of elution buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA pH 8.0, 1% SDS) and incubated at 65C for 15 min to elute. To purify eluted DNA, RNA was degraded by incubation with 2.5ul of 33 mg/mL RNase A (Thermo, #EN0531) at 37C for 2h. Protein was degraded by incubation of 10ul of 20 mg/mL proteinase K (Invitrogen, #25530049) at 55C for 45 min. Samples were then incubated at 65C for 5h to reverse cross-links. Phenol: chloroform: isoamyl alcohol extraction was performed followed by an ethanol precipitation. The DNA was then resuspended in TE buffer. Tagmentation of ChIP DNA was performed using the Tn5 (Vazyme, #TD501). First, 5ul of streptavidin T1 magnetic beads (Invitrogen, #65601) was washed with 1ml of tween wash buffer (5 mM Tris-HCl pH 7.5, 0.5 mM EDTA pH 8.0, 1 M NaCl, 0.05% Tween-20) and resuspended in 10ul of 2biotin binding buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA pH 8.0, 2 M NaCl). 125 ng purified DNA was added in a total volume of 10ul of water to the beads and incubated at room temperature for 15 min with agitation every 5 min. After capture, beads were separated with a magnet and the supernatant was discarded. Beads were then washed twice with 500ul of tween wash buffer at 55C for 2 min. After washes, beads were resuspended in 1TTBL. Tn5 amount was adjusted linearly for different amounts of post-ChIP DNA, 125 ng of DNA was transposed with 4ul of Tn5. Samples were incubated at 55C with interval shaking for 10 min. Beads were then placed on a magnet, and supernatant was removed. 50 mM EDTA was added to samples and incubated with interval shaking at 50C for 30 min. Samples were washed in 10 mM Tris, pelleted and resuspended for PCR amplification (Vazyme, #TD202). Two biological replicates were performed for each cell line.