GSM5904393: Control Input-replicate B; Homo sapiens; ChIP-Seq
SKMEL-147 cells were crosslinked with 1% PFA (in PBS 1X) for 10 min at RT. The reaction was quenched with 0.125 M glycine for 5 min at room temperature. After washing with PBS, cells were collected and the pellet was lysed in 50 mM Hepes pH7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% Igepal CA-630, and 0.25% Triton X-100, freshly supplemented with 1X Protease Inhibitors Cocktail (Sigma-Aldrich). After centrifugation, isolated nuclei were washed once in 10 mM Tris pH 8.0, 200 mM NaCl, 1 mM EDTA and 0.5 mM EGTA, 1X Protease Inhibitors Cocktail then resuspended in ChIP buffer (10 mM Tris pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% sodium deoxycholate, 0.5% N-Lauryl Sarcosine, 1X Protease Inhibitors Cocktail) prior sonication. After optimization for each cell line, sonication of SKMEL-147 nuclei was carried out for 30 cycles (30 seconds on, 30 seconds off), with high intensity at 4°C in a Bioruptor sonicator (Diagenode).  Under these conditions, chromatin fragments have an average size of 200 bp. Thruplex kit was used according to manufacturer's instructions.