Lightcycler 480 sw 1.5 apparatus

For qChIP assays in cells, Mefs were incubated with 1% formaldehyde/1% paraformaldehyde for 5 min followed by the addition of 125 mM Glycine to stop the reaction. Cells were then washed in phosphate-buffered saline, 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 (epishear, Active motif). qChIPs were carried out by incubating chromatin (Input) with protein G-Dynabeads and the different antibodies: control (irrelevant IgG), affinity-purified rabbit anti-E4F1 polyclonal^{21 (link)}, anti-p53 (1C12, Cell Signaling, 10 μl per IP), anti-H3K4me3 (Cell Signaling, 10 μl per IP), anti-H3H9me3 (Cell Signaling, 10 μl per IP), anti-H3K27me3 (Cell Signaling, 10 μl per IP), anti-H3K18ac (Cell Signaling, 10 μl per IP), anti-H3K27ac (Cell Signaling, 10 μl per IP) antibodies. After overnight 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 a percentage of the input. Primers used for qChIP assays are provided in supplementary table 1.

Total RNA was isolated from cell lines lysed in TRIzol (Invitrogen, Fisher Scientific, Illkirch-Graffenstaden, France), while PDX tumors were lysed using Lysing Matrix D (MP Biomedicals™, Doornveld, France). Subsequently, the RNA was extracted using the RNeasy Kit (Qiagen, Les Ulis, France) following manufacturer instructions. cDNAs were synthesized from 1μg of total RNAs using random hexamers and SuperScript III Reverse transcription (Invitrogen, Fisher Scientific, Illkirch-Graffenstaden, France). Real-time qPCR was performed on a LightCycler 480 SW 1.5 apparatus (Roche, Meylan, France) with ONEGreen^® FAST QPCR PREMIX (Ozyme, Saint Cyr l’Ecole, France) and designed human specific primers (Supplementary Table 1). Results were quantified with a standard curve generated by serial dilutions of a reference cDNA preparation. GAPDH transcripts were used for normalization. The fold change in gene expression was calculated as: Fold change = 2^-ΔΔCT.

To perform chip assays 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, 0.5 mM EGTA, 0.1% SDS, 0.5% Triton X-100, 0.05% NaDoc and protease inhibitors) and chromatin was shared by sonication. qChIPs were carried out by incubating cell lysates (Input) with 20 μL of protein G-Dynabeads and 5 ug of antibody. The same amount of rabbit IgGs (Santa Cruz, Boulogne Billancourt, France) 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, Boulogne Billancourt, France). Results are represented as the mean value of at least three 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.