Illumina HiSeq 1500 sequencing; RNAseq of mouse embryonic fibroblasts from conditional knock-in mice carrying a phosphorylation-deficient S180A (SA) mutation in exon 5 of the endogenous mouse Trp53 gene locus
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). MEFs were treated for 6.5 hours with either 10µM Nutlin3a in DMSO or DMSO (10µl) alone.  Total RNAs were purified using the RNeasy kit and the Qiacube robot from Qiagen. The quality of RNAs and the p53-specific induction of p21 expression upon Nutlin3a treatment has been verified by Reverse Transcription - quantitative Polymerase Chain Reaction (RTqPCR) using the Superscript VILO cDNA synthesis kit from Invitrogen and the Absolute qPCR cybergreen mix from Thermo Scientific, respectively. qPCR was performed on the Lightcycler 480 from Roche. RNASeq libraries were prepared from total RNA using the TruSeq Stranded mRNA LT kit (Illumina) according to the manufacturer's instructions.