GSM1843241: 99-In (7477); Homo sapiens; ChIP-Seq
Five million human melanocyte cells per ChIP reaction were cross-linked and lysed by incubation in lysis buffer containing 50 mM Hepes pH 7.9, 140 mM NaCl, 1 mM EDTA, 0.5% NP-40, 0.25 % Triton X-100, 10% glycerol, and 1x complete protease inhibitor cocktail (Roche) for 20 min on ice. After washing twice with cold washing buffer (10 mM Tris-HCl pH 8.1, 200 mM NaCl, 1 mM EDTA, and 0.5 mM EGTA), the nuclear pellet was resuspended in 1 ml of cold shearing buffer (10 mM Tris-HCl pH 8.1, 1 mM EDTA, and 0.1% SDS) and then sheared to produce chromatin fragments with a length distribution of 100 to 300 bp using a Covaris S220 sonicator (Covaris) with the following settings: 12.5 min, 5% duty cycle, 140 Watts peak incident power, and 200 cycles per burst. After adding 1% Triton X-100 and 150 mM NaCl (final concentrations) to the sheared chromatin and mixing, the solution was cleared by centrifugation at 14,000xg for 10 min at 4°C. Chromatin fragments were incubated with 8 μg of polyclonal anti-H3K27me3 antibody (07-449; Millipore; Billerica, MA) overnight at 4°C with rotation, and then immunoprecipitated using Dynabeads Protein G (Invitrogen; Carlsbad, CA). ChIP DNA fragments were end-repaired using NEB End Repair Enzyme mix (NEB) and A-tailed using NEB Klenow Fragment (3’→5’ exo-). After Illumina’s adapter ligation, DNA was PCR amplified with Illumina primers for 12 cycles, and the library fragments of 200 bp to 500 bp were size selected by using Ampure XP beads (Beckman Coulter; Brea, CA) according to the manufacturer’s instructions.