Cells were crosslinked with 1% formaldehyde diluted in PBS, without the addition of other co-crosslinkers, for 5 min at room temperature.  After glycin quenching, the cell pellets were lysed in buffer containing  50 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton, 0.1% Na-Deoxycholate, 0.1% SDS, supplemented with complete protease inhibitor cocktail (Thermo Scientific), and sonicated with Covaris sonicator, resulting in chromatin fragments of 250 bp average size.  The supernatant was diluted 10 times with the above buffer without SDS, and subjected to immunoprecipitations with 2 ug of antibody or control IgG conjugated with Dynabeads Protein A or G (Invitrogen) at 4 °C for overnight.  The beads were then washed 5 times with buffer containing 50 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton, and 1 time with final wash buffer (50 mM Tris, pH 8.0, 10 mM EDTA, 50 mM NaCl), followed by elution with incubation of elution buffer (final wash buffer plus 1% SDS) at 65 °C for 30 min with agitation in a thermomixer. The ChIP and input were purified and used for qPCR analysis or for constructing sequencing libraries with the NEBNext Ultra kit (New England Biolabs).
GEO Accession
GSM1827557