ChIP-Seq Protocol for Chromatin Analysis

ChIP assays were performed from approximately 10⁷ cells per experiment, according to previously described protocol with slight modifications [17] (link). Briefly, cells were crosslinked with 1% formaldehyde for 10 min at room temperature and formaldehyde was quenched by addition of glycine to a final concentration of 0.125 M. Chromatin was sonicated to an average size of 0.5–2 kb, using Bioruptor (Diagenode). 50–75 μL of protein G dynal beads (Invitrogen) were used to capture 3–5 μg of antibody in phosphate citrate buffer pH = 5.0 (2.4 mM citric acid, 5.16 mM Na₂HPO₄) for 30 min at 27 °C. Antibody bead complexes were rinsed 2x with PBS and added to sonicated chromatin and rotated at 4 °C overnight. 10% of chromatin was reserved as “input” DNA. Magnetic beads were washed and chromatin eluted, followed by reversal of the crosslinkings and DNA purification. Resultant ChIP DNA was dissolved in TE. Results were verified with qPCR of 7 selected regions [17] (link).