GSM8192535: ChIPseq_501mel shNIPBL_#2 6D MITF; Homo sapiens; ChIP-Seq
20 x 106 cells were harvested and fixed in 1% formaldehyde for 10 min with gentle shaking. Glycine was added to final concentration of 125 mM and incubated for 5 min. Cells were washed once with PBS, resuspended in LB1 [50mM HEPES (pH 7.5), 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100 and 1× complete EDTA-free protease inhibitor] and incubated with rotation at 4 °C for 10min. Samples were pelleted, resuspended in LB2 [10mM Tris-HCl (pH 8.0), 200mM NaCl, 1mM EDTA, 0.5mM EGTA and 1× complete protease inhibitor] and incubated rotating at 4 °C for 10min. Nuclei were pelleted, resuspended in LB3 with 0.5% SDS [0.5% SDS, 20mM Tris-HCl (pH 8.0), 150mM NaCl, 2mM EDTA, 1% Triton X-100 and 1× complete protease inhibitor] and sonicated using Covaris E220 focused ultrasonicator (peak power = 150 W, duty factor = 2%, cycle per burst = 200) for 12 min. Soluble chromatin extracts were cleared by centrifugation and 1% was kept as input DNA. Sheared chromatin was diluted to contain 0.16% SDS for antibody binding and incubated overnight at 4 °C with 2 ug anti-SMC1A (Bethyl Laboratories Inc, A300-055A), anti-MITF (Active Motif, 91201) or anti-CTCF (Cell Signlaing Technology, 3418S). Spike-in chromatin (Active motif, 53083) and antibody (Active motif, 61686) were included to normalize SMC1A ChIP-seq.  20 ul Pierce ChIP-grade magnetic beads were used for precipitation at 4 °C for 6 hr. Beads were washed 3x LiCl buffer [250 mM LiCl, 10 mM Tris-HCl (pH 8.0), 1% sodium deoxycholate, 1% NP-40 and 1 mM EDTA], 1x RIPA wash buffer [500 mM LiCl, 50 mM HEPES-KOH (pH 7.6), 0.7% sodium deoxycholate, 1% NP-40 and 1 mM EDTA] and 1x TE [10 mM Tris-HCl (pH 8.0) and 1 mM EDTA] + 50 mM NaCl. Chromatin was eluted in elution buffer [1% SDS, 50 mM Tris-HCl (pH 8.0) and 10 mM EDTA] and incubated at 65 °C for 30 min. Eluate was reverse cross-linked at 65 °C overnight, treated with RNaseA (Roche) at 37 °C for 1 hr and Proteinase K (Roche) at 55 °C for 2 hr before recovered using a miniElute PCR purification kit from Qiagen. Library was prepared using KAPA HTP library kit (KAPA biosystems). ChIP libraries were sequenced to obtain 30-40 M reads for each sample on NovaSeq 6000 with PE50.