mM EDTA (pH 8), 2% (v/v) Triton X, 0.2% (v/v) Na deoxycholate, 10% glycerol] similarly supplemented as described above. Sonication to fragment cross-linked chromatin was carried out using the Bioruptor (Diagenode) at high power over 12 cycles of 30 s each. Fragmented chromatin was incubated overnight with Dynabeads (Life Technologies) precoated with 10 μg of anti-IRF2 antibody [IRF-2 (C-19): sc-498, Santa Cruz Biotechnology]. Progressive washing of the chromatin-Dynabead mixture was then carried out in 10 mM tris (pH 8), 1 mM EDTA (pH 8), 1% (v/v) NP-40, 0.7% (v/v) Na deoxycholate, 0.5 M LiCl, supplemented as previously but using only 0.5× Complete Protease Inhibitor. Cross-links were reversed after an overnight incubation in elution buffer [10 mM tris (pH 8), 1 mM EDTA (pH 8), and 1% SDS]. Immunoprecipitated DNA was obtained using phenol-chloroform extraction and purified after precipitation in ethanol. DNA was quantified using the High Sensitivity Qubit system (Life Technologies), and the fragmentation profile was assessed using a DNA HS 2200 TapeStation chip (Agilent).
