Mus musculus
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.
ENA-FIRST-PUBLIC
2020-09-08T17:06:07Z
ENA-LAST-UPDATE
2020-06-03T09:46:13Z
External Id
SAMEA6871715
INSDC center name
Institute of Molecular Oncology, Philipps-University Marburg
INSDC first public
2020-09-08T17:06:07Z
INSDC last update
2020-06-03T09:46:13Z
INSDC status
public
Submitter Id
E-MTAB-9170:SA_Nutlin
age
12.5 to 13.5
broker name
ArrayExpress
cell type
mouse embryonic fibroblast cell
common name
house mouse
developmental stage
embryo stage
genotype
Trp S180A
organism part
mouse embryonic fibroblast cell
sample name
E-MTAB-9170:SA_Nutlin
scientific_name
Mus musculus
sex
male
strain
B6.129S