GSM9063531: Exp.2_SET2_input_chromatin; Homo sapiens; ChIP-Seq
Cells were double cross-linked with 50 µL/mL of 40 mM disuccinimidyl glutarate (DSG) for 30 minutes and 2% formaldehyde solution for 10 minutes. The reaction was quenched by adding 200 µL of 1.25 M glycine and 100 µL of 1 M Tris-HCl per mL of suspension. Cells were pelleted by centrifugation (1400 rpm, 5 min). Cells were lysed in 1 mL of Cytoplasmic Lysis Buffer (20 mM Tris-HCl pH 7.5, 300 mM NaCl, 2 mM EDTA, 0.5% NP-40, 1% Triton X-100; freshly supplemented with 1 mM PMSF and cOmplete™ Protease Inhibitor Cocktail). Nuclei were extracted by applying 10–15 strokes with a pre-chilled Dounce homogenizer. The nuclear pellet was resuspended in Sonication Buffer (10 mM Tris-HCl pH 8.1, 100 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.5% N-lauroylsarcosine, 0.1% SDS; freshly supplemented with 1 mM PMSF and cOmplete™ Protease Inhibitor Cocktail). Chromatin was sonicated to an average fragment size of 200–600 bp using a Covaris ME220 Focused-ultrasonicator. For immunoprecipitation, 5 µg of antibody per IP were used. Beads were then resuspended in Wash Buffer (50 mM HEPES pH 7.6, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% sodium deoxycholate; freshly supplemented with 1 mM PMSF) and washed six times with 800 µL Wash Buffer by gentle inversion for 2 minutes per wash, followed by a final wash with 1 mL of 1x TE buffer. Chromatin was eluted in 150 µL Elution Buffer and decrosslinked at 65°C overnight. Library preparation for Illumina sequencing was performed using the NEBNext Ultra II DNA Library Prep Kit, following the manufacturer's instructions. Libraries were purified with 0.9× AmpureXP beads