Rabbit iggs

ChIP assays were mainly performed as previously described (Riscal et al., 2016 (link)), with some modifications. Briefly, 10⁷ cells were cross-linked in 1% formaldehyde/1% paraformaldehyde for 5 min, followed by addition of 125 mM Glycine to stop the reaction. Cells were then washed in PBS, resuspended in lysis buffer (10 mM Tris pH 8,140 mM NaCl, 0.1% SDS, 0.5% Triton X-100, 0.05% NaDoc, 1 mM EDTA, 0.5 mM EGTA and protease inhibitors) and chromatin was sheared by sonication. qChIPs were carried out by incubating cell lysates (Input) with 20 μL of protein G-Dynabeads and 5 μg of antibody (MDM2, clone N20, Santa Cruz; TFAM, Cell Signaling). The same amount of rabbit IgGs (Santa Cruz) was used for control ChIP experiments. After O/N incubation, washing, reverse cross-linking and treatment with both RNase A and Proteinase K, proteins were removed with phenol/chloroform extraction and DNA was recovered using the NucleoSpin Extract II kit. Input and immunoprecipitated DNA were then analyzed by QPCR using the SYBR Green Master mix on a LightCycler 480 SW 1.5 apparatus (Roche). Results are represented as the mean value of at least 3 independent experiments of immunoprecipitated chromatin (calculated as a percentage of the input) with the indicated antibodies after normalization by a control ChIP performed with rabbit IgGs.