GSM7325032: HiChIP_H3K27ac_SKMEL-23 rep 2; Homo sapiens; OTHER
15 x 106 cells were cross-linked with 1% formaldehyde for 10 min. Fixation was quenched with 125 mM glycine. Cells were washed once with PBS and lysed for 30 min using in situ Hi-C lysis buffer [10mM Tris-HCl (pH 7.5), 10mM NaCl, 0.2% NP-40 and 1x complete EDTA-free protease inhibitors (Roche)]. Nuclei were treated with 0.5% SDS followed by 1.25% Triton X-100 before digesting with 400 U MboI (NEB) for 4 hr. Restriction enzyme digested overhangs were filled in with biotin-dATP (Thermo fisher scientific) and ligated at 16 °C overnight. Nuclei were sonicated in nuclear lysis buffer [50mM Tris-HCl (pH 7.5), 10mM EDTA, 0.5% SDS and 1x EDTA-free protease inhibitor (Roche)] using Covaris E220 with the following parameter: peak power = 140 W, duty factor = 5%, cycle per burst = 200 and time = 4 min. Pull-down with anti-H3K27ac antibody (Abcam, ab4729) was performed by diluting sheared chromatin to 0.16% SDS in buffer with Pierce ChIP-grade magnetic beads. ChIP reaction was washed 3x LiCl buffer [250mM LiCl, 10mM Tris-HCl (pH 8.0), 1% sodium deoxycholate, 1% NP-40 and 1mM EDTA], 1x RIPA wash buffer [500mM LiCl, 50mM HEPES-KOH (pH 7.6), 0.7% sodium deoxycholate, 1% NP-40 and 1mM EDTA] and 1x TE [10mM Tris-HCl (pH 8.0) and 1mM EDTA] + 50 mM NaCl. Chromatin was eluted in 50 mM sodium bicarbonate (pH 8.0) with 1% SDS and reverse cross-linked with proteinase K at 65'C overnight. Streptavidin C1 beads (Thermo fisher scientific) were used to capture biotinylated DNA. Beads were washed twice with tween wash buffer [5 mM Tris-HCl (pH 7.5), 0.5mM EDTA. 1M NaCl and 0.05% Tween 20]. To build library for Illumina NovaSeq 6000 sequencing, Tn5 (Illumina) was used for tagmentation and amplified with Nextera Ad1_noMX and Ad2.X primers using 2x Phusion high-fidelity PCR mix (NEB). AMPure beads (Beckman coulter) were used for double-sided size selection (0.5x and 1x of the PCR volume) and purification of library DNA sequenced with PE75 500M reads.