|
| Status |
Public on Feb 01, 2024 |
| Title |
WI38_RNA_PHARM_INHIB_RAS_D07_REP2 |
| Sample type |
RNA |
| |
|
| Source name |
WI38, RASOIS, D07
|
| Organism |
Homo sapiens |
| Characteristics |
treatment: RAS-Oncogene Induced Senescence
timepoint: D07
|
| Treatment protocol |
DNA damage-induced senescence (DDIS) was triggered by etoposide treatment at a concentration of 20µM for two 2 days; cells were then washed and incubated with fresh medium without drug. For the matching samples, Rapamycin was added to fresh medium at a final concentration of 20 nM just before use. Cells were collected and processed at the indicated time points following the treatment. RAS-OIS was induced by adding 400 nM 4-hydroxytamoxifen (4-OHT) to the culture medium, and DMOG was added to fresh medium at a final concentration of 1 mM in the corresponding samples. As before, cells were collected and processed at the indicated time points following the treatment.
|
| Growth protocol |
WI-38 fibroblasts (purchased from ATCC) were cultured in DMEM GLUTAMAX, high glucose (Gibco) medium containing 10% fetal bovine serum (FBS), and 1x PenStrep (Thermofischer) in a humidified incubator at 37 °C with a 5% atmospheric concentration of O2 and CO2. The medium was changed every 2 days. Cells were split as they reached a confluency of 70-80%.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Hybridization cocktail of fragmented and labeled ss-cDNA were incubated 16hr, 60rpm at 45°C on the Human Transcriptome Arrays 2.0. Genechips were washed and stained in the Affymetrix Fluidics Station 450 according to the standard GeneChip® Expression Wash, Stain, and Scan User Manual for Cartridge Arrays (PN 702731)
|
| Label |
biotin
|
| Label protocol |
Fragmented and labeled DNA targets were prepared according to the standard Affymetrix WT PLUS Reagent Kit protocol from 100 ng total RNA starting material and 5.5 µg single strand cDNA.
|
| |
|
| Hybridization protocol |
Hybridization cocktail of fragmented and labeled ss-cDNA were incubated 16hr, 60rpm at 45°C on the Human Transcriptome Arrays 2.0. Genechips were washed and stained in the Affymetrix Fluidics Station 450 according to the standard GeneChip® Expression Wash, Stain, and Scan User Manual for Cartridge Arrays (PN 702731)
|
| Scan protocol |
GeneChips were scanned using the Affymetrix GCS 3000 scanner.
|
| Data processing |
Raw Affymetrix HTA 2.0 array intensity data were analyzed using open-source Bioconductor packages on R. Internal control probe sets were removed and average expression deciles over time-points were then defined independently for each treatment. Probes whose average expression was lower athan the 4th expression decile in both conditions were removed for subsequent analyses.
|
| |
|
| Submission date |
Nov 28, 2023 |
| Last update date |
Feb 01, 2024 |
| Contact name |
José Américo Nabuco Leva Ferreira Freitas |
| Organization name |
Institut Mondor de Recherche Biomedicale
|
| Lab |
SENESCENCE, METABOLISM AND CARDIOVASCULAR DISEASES
|
| Street address |
8 rue du Général Sarrail
|
| City |
CRETEIL |
| ZIP/Postal code |
94010 |
| Country |
France |
| |
|
| Platform ID |
GPL17586 |
| Series (2) |
| GSE248823 |
Glycerol-3-phosphate and Phosphoethanolamine homeostatic switch triggers senescence by rewiring lipid metabolism II |
| GSE248824 |
Glycerol-3-phosphate and Phosphoethanolamine homeostatic switch triggers senescence by rewiring lipid metabolism |
|
