GSM6430040: A375_H3K27ac_HiChIP; Homo sapiens; ChIP-Seq
A375 cells were counted, and ~1.5 × 107 cells were crosslinked with 1% formaldehyde in phosphate-buffered saline (PBS; ThermoFisher, 10010023) at room temperature for 10 min, and then quenched with 0.125 M Glycine (Sigma-Aldrich, 50046) on ice for 5 min. Next, crosslinked cells were lysed in Hi-C lysis buffer (10mM Tris-HCl pH8.0 [ThermoFisher, 15568025], 10mM NaCl [Sigma-Aldrich, S7653], and 0.2% Igepal CA630 [Sigma-Aldrich, I3021]) with 50 μl of protease inhibitors (Sigma, P8340), and nuclei were extracted and digested with MboI restriction enzyme (NEB, R0147) for 2 h. After digestion, nuclei were resuspended in NEB buffer supplemented with DNA polymerase I, Large (Klenow) fragment (NEB, M0210) to fill in the restriction fragment overhangs with Biotin-14-dATP (Life Technologies，19524-016) for 1 h. Proximal ligation was performed with T4 DNA ligase (NEB, M0202M) for 4 h at 16°C and nuclei were harvested. The nuclei were transferred to shear chromatin using a Biorupter system (Diagenode, B01060001). The sheared chromatin was incubated overnight with 8 μl of H3K27ac antibody (Abcam, ab4729), and then Protein A-agarose beads (Millipore, 16-157) were used to capture H3K27Ac-associated chromatin. The whole H3K27ac-ChIP process was performed with the PierceTM Magnetic ChIP kit (ThermoFisher, 26157), the final DNA was eluted with 15 μl Nuclease-Free Water (ThermoFisher, AM9930). The eluted DNA was sent for DNA biotin pull-down with Dynabeads MyOne Streptavidin T1 beads (Life technologies, 65602). Sixty micrograms of DNA were used for PCR amplification, and then AMPure XP beads (Beckman Coulter, A63880) was used to select a size range of 300~600 bp products.