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| Status |
Public on Apr 05, 2023 |
| Title |
GM21 fibroblasts, RAS, biol rep 1, day 13 |
| Sample type |
RNA |
| |
|
| Source name |
GM21 fibroblasts at day 13 post-transduction with H-RAS-G12V
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: skin fibroblast
|
| Treatment protocol |
GM21-RASV12 fibroblasts and their empty vector counterparts were generated by retroviral transduction. GM21 fibroblasts constitutively expressing non-targeting and POU2F2-targeting shRNAs (pLKO.1-Neo-CMV-tGFP, pLKO.1-Neo-CMV-tGFP-TRCN0000245324 and pLKO.1-Neo-CMV-tGFP-TRCN0000245325) (Millipore-Sigma, Saint Louis, MO) were generated by lentiviral transduction and selected with 400 µg/ml neomycin for seven days. Cells were subsequently retrovirally transduced with RASV12
|
| Growth protocol |
Human somatic GM21 foreskin fibroblasts (Coriell Institute, Camden, NJ) and WI-38 human fetal lung fibroblasts (European Collection of Authenticated Cell Cultures, Porton Down, UK) were cultured in a DMEM medium containing 10% fetal bovine serum (FBS) and 1× penicillin/streptomycin (Corning) at 37 °C in a 2% oxygen atmosphere
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Macherey-Nagel RNA XS columns. At least 200 ng RNA with RIN number 9 were used for library construction.
|
| 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.
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| |
|
| Hybridization protocol |
Hybridization cocktail of fragmented and labeled ss-cDNA were incubated 16hr, 60rpm at 45°C on 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.
|
| Description |
Gene expression profiling of the entry and exit from RAS-induced senescence in GM21 fibroblasts
|
| Data processing |
Raw Affymetrix HTA 2.0 array intensity data were analyzed using open-source Bioconductor packages on R. All the data were normalized all together using the robust multi-array average normalization approach implemented in the oligo package (Carvalho and Irizarry, 2010). Internal control probe sets were removed and average expression deciles over all treatment and time points. Probes whose average expression was lower than the 4th expression decile were removed for subsequent analyses.
To remove sources of experimental variation and consider batch effects, data were finally corrected with the sva package. Principal component analysis and bi-clustering based on Pearson’s correlation and Ward’s aggregation criterion were used to confirm consistency between biological replicates and experimental conditions at each step of the pre-processing.
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| |
|
| Submission date |
Jun 08, 2022 |
| Last update date |
Apr 06, 2023 |
| Contact name |
Ricardo Iván Martínez Zamudio |
| E-mail(s) |
rm1238@rwjms.rutgers.edu
|
| Organization name |
Rutgers Biomedical and Health Sciences | Rutgers-Robert Wood Johnson Medical School
|
| Department |
Pharmacology
|
| Lab |
Martínez Zamudio Lab
|
| Street address |
675 Hoes Lane West
|
| City |
Piscataway |
| State/province |
New Jersey |
| ZIP/Postal code |
08854 |
| Country |
USA |
| |
|
| Platform ID |
GPL17586 |
| Series (2) |
| GSE205692 |
Escape From Oncogene-Induced Senescence is Controlled by POU2F2 and Memorized by Chromatin Scars [Expression] |
| GSE206496 |
Escape From Oncogene-Induced Senescence is Controlled by POU2F2 and Memorized by Chromatin Scars |
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