GSM9069598: U-2 OS parental cell line ChIP, normal rabbit IgG control; Homo sapiens; ChIP-Seq
Cells were washed twice with ice-cold PBS, scraped, centrifuged for 10 min at 4000 rpm, resuspended in cell lysis buffer (5 mM PIPES, pH 8.0, 85 mM KCl, and 0.5% NP-40, 1 ml/15 cm plate) and incubated for 10 min on ice. During the incubation, the lysates were repeatedly pipetted up and down every 5 minutes. Lysates were then centrifuged for 10 min at 4000 rpm. Nuclear pellets were resuspended in 6 volumes of sonication buffer (50 mM Tris-HCl, pH 8.1, 10 mM EDTA, 0.1% SDS), incubated on ice for 10 min, and sonicated to obtain DNA fragments below 2000 bp in length (Covaris S220 sonicator, 20% Duty factor, 200 cycles/burst, 100 peak incident power, 11 cycles of 30" on and 30" off). Sonicated lysates were cleared by centrifugation and 500 μg of chromatin was diluted in RIPA buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% SDS, 0.1% Na-deoxycholate, 140 mM NaCl) to a final concentration of 0.8 μg/μl, precleared with Protein G Dynabeads (Fisher 1009D) for 2 hours at 4°C and immunoprecipitated overnight with 1.25 μg of normal rabbit IgGs or anti-CTCF antibodies. 10% of the precleared chromatin was saved as input. Immunoprecipitated DNA was purified with the Qiagen QIAquick PCR Purification Kit, eluted in 36 μl of water, 25 ul of which were used used for library prep. NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (using half of the recommended volumes); adaptors diluted 1:10; 9 PCR cycles; 20 ng starting material for inputs; 25 ul for CTCF ChIP samples.