title: SA_DMSO
organism: Mus musculus
description: Protocols: Primary MEFs were isolated from E12.5-13.5 mouse embryos and amplified under low oxygen conditions (3% O2). All cells were maintained at ambient oxygen in humidified cell culture incubator (37°C with 5% CO2) in high-glucose Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich), 100 U/ml penicillin and 100 µg/ml streptomycin (Life technologies). Primary MEFs were treated for 16 hours with 10 µM nutlin-3a-3a or 0,1% DMSO as control. Cells were fixed on plates with 0.88% paraformaldehyde (PFA) for 10 min at room temperature and quenched by adding glycine to a final concentration of 54 mM for 5 min. Cells were washed twice with ice-cold PBS and scraped off the plate with PBS supplemented with proteinase-inhibitors (Roche). Cell pellets were lysed in SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.1) supplemented with protease-inhibitors (2x107 cells/ml lysis buffer) and sonicated using the X Bioruptor® Sonication System (Diagenode) to obtain 300-1000 bp DNA fragments. The shearing efficiency was controlled using agarose gel electrophoresis. After centrifugation (10000 g, 10 min, 20°C), 100 µl sheared chromatin was diluted 1:10 with dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8.1, 167 mM NaCl) and pre-cleared for 1 h with 50 µl Protein G sepharose (50% slurry in 20% ethanol) beads (GE Healthcare) at 4°C. The p53 protein was immunoprecipitated overnight at 4°C with 2.5 µg anti-p53 antibody (FL393, Santa Cruz), normal rabbit IgG (Santa Cruz) was used as the control. The protein complexes were pulled down for 4 hours at 4°C with 50 µl Protein G sepharose beads. Beads were washed with Low Salt Immune Complex Wash Buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 150 mM NaCl), then with High Salt Immune Complex Wash Buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 500 mM NaCl), with LiCl Immune Complex Wash Buffer (0.25 M LiCl, 1% IGEPAL-CA630, 1% deoxycholic acid (sodium salt), 2 mM EDTA, 10 mM Tris-HCl pH 8.1), and finally twice with TE (10 mM Tris-HCl pH 8.1, 1 mM EDTA). Protein/DNA crosslinks were eluted twice for 15 min at 20°C in 100 µl elution buffer (0.1 M NaHCO3, 1% SDS). 1% input stored at -20°C was treated in the same way. Crosslinking was reverted upon overnight incubation at 65°C in elution buffer supplemented with 200 mM NaCl followed by RNaseA digestion at 37°C for 30 min, addition of 40 mM Tris-HCl pH 6.5 and 10 mM EDTA, proteinase K digestion for 2 hours at 55°C and inactivation of proteinase K at 99°C for 10 min. DNA was purified using the QIAquick PCR Purification Kit (Qiagen). ChIP-seq libraries were prepared from purified ChIP DNA with the Microplex Library Preparation Kit (Diagenode) according to the manufacturer's instructions.