description: Protocols: Cells where washed in PBS buffer and trypsininated with 2% Trypsin/PBS, then resuspended in high-glucose Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich). Cell suspensions were centrifuged for 5 minutes at 300g and washed in PBS, then centirfuged again. Cell pellets were resuspended in 600 µl RLT buffer (QIAGEN). The Saos-2 human osteosarcoma cell line was obtained from the American Tissue Collection Center (ATCC) and authenticated by short tandem repeat analysis at the Leibniz Institute DSMZ – German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany. For experiments, all cells were maintained at ambient oxygen in a humidified cell culture incubator (37°C, 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). Recombinant adenoviral vectors co-expressing GFP and FLAG-tagged p53 mutants were produced in Ad293 cells (Agilent) using the pAdEasy system (Agilent) and used for infection of Saos-2 cells as described before (Schlereth et al., 2010). Ad-vectors were titrated to precisely regulate the amount of ectopically expressed p53, while equalizing the total amount of Ad-vector encoding GFP only, using Western blotting and GFP flow cytometry. RNA was isolated using the RNeasy Mini kit (Qiagen) and RNA quality was assessed using the Experion RNA StdSens Analysis Kit (Bio‐Rad). RNA‐seq libraries were prepared from total RNA using the QuantSeq 3′ mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen) in combination with the UMI Second Strand Synthesis Module for QuantSeq FWD (Illumina, Read 1) (Lexogen) according to the manufacturer's instructions. Quality of sequencing libraries was controlled on a Bioanalyzer 2100 using the Agilent High Sensitivity DNA Kit (Agilent).
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2022-04-26
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2022-04-26
attributes.tag: External Id
attributes.value: SAMEA8235613
attributes.tag: INSDC center alias
attributes.value: Institute of Molecular Oncology, Philipps-University Marburg
attributes.tag: INSDC center name
attributes.value: Institute of Molecular Oncology, Philipps-University Marburg
attributes.tag: INSDC first public
attributes.value: 2022-04-26T16:15:52Z
attributes.tag: INSDC last update
attributes.value: 2022-04-26T16:15:52Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-10245:NM-GFP-2
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell_line
attributes.value: SaOS-2
attributes.tag: common name
attributes.value: human
attributes.tag: developmental stage
attributes.value: infant
attributes.tag: disease
attributes.value: osteosarcoma
attributes.tag: genotype
attributes.value: p53 -/-
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: organism part
attributes.value: bone
attributes.tag: sample name
attributes.value: E-MTAB-10245:NM-GFP-2
attributes.tag: sex
attributes.value: female