organism: Mus musculus
description: 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.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2019-04-18T04:02:26Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-04-26T16:41:01Z
attributes.tag: External Id
attributes.value: SAMEA4613287
attributes.tag: INSDC center name
attributes.value: Institute of Molecular Oncology, Philipps-University Marburg
attributes.tag: INSDC first public
attributes.value: 2019-04-18T04:02:26Z
attributes.tag: INSDC last update
attributes.value: 2018-04-26T16:41:01Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-6774:p53_EE_1E2_Nutlin
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: mouse embryonic fibroblast cell
attributes.tag: common name
attributes.value: house mouse
attributes.tag: developmental stage
attributes.value: embryonic day 12.5 to 14.5
attributes.tag: genotype
attributes.value: Tg(Prm-cre)58Og/J; Trp53 Arg178Asp
attributes.tag: organism part
attributes.value: embryo
attributes.tag: sample name
attributes.value: E-MTAB-6774:p53_EE_1E2_Nutlin
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: strain
attributes.value: 129/Sv