|
| Status |
Public on Feb 01, 2024 |
| Title |
MYOBLAST_SENESCENCE_TRANSCRIPTOME_RAS_96H_REP1 |
| Sample type |
RNA |
| |
|
| Source name |
SkMC, RASOIS, D04
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: SkMC
treatment: RAS-Oncogene Induced Senescence
timepoint: D04
|
| Treatment protocol |
WI-38-ER:RASV12 fibroblasts were generated by retroviral transduction. RAS-OIS was induced by adding 400 nM 4-hydroxytamoxifen (4-OHT) to the culture medium. Cells were collected and processed at the indicated time points following the treatment. 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. Cells were collected and processed at the indicated time points following the treatment. Replicative senescence was obtained through proliferative exhaustion.
|
| 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%. Primary human myoblasts (SkMC) were isolated from a skeletal muscle biopsy of a healthy donor (PromoCell #C-12530, Lot 414Z025.11) and were purified with an immuno-magnetic sorting system using CD56/NCAM magnetic beads (Miltenyui Biotec #130-050-401) following the manufacturer’s specifications. The purified CD56-positive myoblasts were seeded in cell culture dishes coated with type I collagen (Sigma-Aldrich #C 8919) and cultured in the proliferation medium (DMEM-high glucose (Sigma # D6429), 20% FBS (Life Technologies #10270106), 50µg/ml gentamicin (Life technologies # 15750037), 0,5% Ultroser G (PALL # 15950-017) at 37°C with 5% CO2.
|
| 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.
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| |
|
| Submission date |
Nov 28, 2023 |
| Last update date |
Feb 06, 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) |
| GSE248822 |
Glycerol-3-phosphate and Phosphoethanolamine homeostatic switch triggers senescence by rewiring lipid metabolism I |
| GSE248824 |
Glycerol-3-phosphate and Phosphoethanolamine homeostatic switch triggers senescence by rewiring lipid metabolism |
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