p53_EE_1E5_DMSO
Mus musculus
Protocols: 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). 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.