Cells were harvested, washed with Episerf (LifeTechnologies, #10732), fixed in 10mL per 1x107 cells (10 min in 1% formaldehyde), quenched with TrisHCl in 50mL (at 250 mM final), washed with PBS, and pelleted. Each pellet containing 1x107 cells was lysed, resuspended in 1 mL of sonication buffer on ice (10 mM Tris at pH 8, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% NaDOC, 0.25% NLS, and protease inhibitors), transferred to TC 12x12 tubes (Covaris), and sonicated (Covaris settings: 20 min, 5% duty cycle, 140W, 200 cycles). Sonication was assessed by reverse cross-linking (65°C, RNAse A at 1μg/μL, overnight), followed by DNA extraction. Fragment size (between 200-400bp) was checked on a Bioanalyzer (Agilent 2100). Immunoprecipitations were performed with chromatin from 1x107 cells with Dynabeads (ThermoFisher) in IP buffer (16.25 mM Tris at pH 8.1, 137.5 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1.25% Triton X-100, and protease inhibitors) overnight. Chromatin was reversed cross-linked (65°C, Proteinase K at 400ng/μL, overnight) and DNA was further extracted for analysis. Paired-end libraries were prepared and sequenced in 75bp reads runs according to Illumina instructions.
GEO Accession
GSM2522690