Illumina HiSeq 1500 sequencing; ChipSeq of conditional knock-in mouse carrying the R178E (EE) mutation in exon 5 of the endogenous mouse Trp53 gene locus.
ChipSeq of conditional knock-in mouse carrying the R178E (EE) mutation in exon 5 of the endogenous mouse Trp53 gene locus.
Primary mouse embryonic fibroblasts (MEFs) expressing wild type p53 where isolated from three mouse embryos. MEFs expressing mutant p53(Arg178Asp) (so-called EE mutant of p53) where isolated from four embryos and three embryos led to the isolation of three cell preparations that do not express any p53 (p53 KO). The MEFs were kept in culture as triplicates (13x3=39 samples)until they reached near confluence. Primary MEFs were isolated from E12-14.5 mouse embryos using standard protocols and amplified at low oxygen conditions (5% O2).  Primary MEFs were treated for 16 hours with 10 µM nutlin-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.
Experimental Factor: immunoprecipitate
anti-Trp53 (FL393, Santa Cruz)
Experimental Factor: compound
Nutlin-3a
Experimental Factor: dose
10
Experimental Factor: phenotype
wild type phenotype