GSM8693842: INPUT_rep2; Homo sapiens; ChIP-Seq
Akata cells were crosslinked with a 1% formaldehyde solution in growth medium and then quenched with 2.5M glycine in distilled water. The cells were washed with ice-cold PBS and lysed in 1% SDS lysis buffer (50 mM Tris, 10 mM EDTA, and 1% SDS) supplemented with 1X cOmplete EDTA-free protease inhibitor. Chromatin was sheared to 100-300 bp with Bioruptor Pico sonication, followed by centrifugation at 15,000 x g for 10 minutes at 4 °C. The resulting supernatant was diluted at a 1:10 ratio in ChIP dilution buffer (1.2 mM EDTA, 16.7 mM Tris, 167 mM NaCl, 0.01% SDS, and 1.1% Triton X-100) supplemented with 1X cOmplete EDTA-free protease inhibitor. 1% of the sheared chromatin was saved as Input and stored at -80 °C until use. The diluted chromatin was rotated overnight at 4 °C with antibody and 20 μl protein A/G magnetic beads. Next day, the immunocomplexes were subsequently washed twice with low-salt buffer (150 mM NaCl, 2 mM EDTA, 20 mM Tris-HCl, 0.1% SDS, 1% Triton X-100) and high-salt buffer (500 mM NaCl, 2 mM EDTA, 20 mM Tris-HCl, 0.1% SDS, 1% Triton X-100), and once with LiCl buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris) and TE buffer (1 mM EDTA, 10 mM Tris). After eluting in Elution buffer (100 mM NaHCO3, 1% SDS), DNA was reverse cross-linked for 2 hours at 65 °C. Immunoprecipitated DNA was purified with QIAquick PCR purification kit. Immunoprecipitated DNA were subjected to library preparation using NEBNext Ultra DNA Library Prep Kit