Chromatin Immunoprecipitation Profiling of Osteoclast Precursors

ChIP was performed as previously described ^{14 (link)}. Briefly, 2 × 10⁷ human primary OCPs were fixed by adding formaldehyde directly to the medium to a final concentration of 1 % for 5 min. Cells were harvested, washed, and lysed. Chromatin was sheared by sonication to a length of approximately 500 base pairs using a Bioruptor sonicator (Diagenode). Sheared chromatin was precleared and then immunoprecipitated with antibodies; c-myc (Abcam; ab56), Brd4 (Bethyl Lab; A301-985A), pan acetyl-H4 (Millipore; 06-866), and CBP (Abcam; ab2832). Immune complexes were subsequently collected and washed, and DNA crosslinking was reversed by heating at 65°C overnight. Following proteinase K digestion, DNA was recovered using the PCR purification kit (Qiagen) and real time PCR was performed to detect the occupancy of target proteins. Signals obtained from the ChIP are divided by signals obtained from an input sample. This input sample represents the amount of chromatin used in the ChIP. The primer sequences are listed in Supplementary Table 3.