|
| Status |
Public on Jun 21, 2019 |
| Title |
Non-targeting shRNA replicate 1 |
| Sample type |
RNA |
| |
|
| Source name |
A375 melanoma cells transduced with non-targeting shRNA
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: A375
cell type: melanoma cells
shRNA: Non-targeting (shLUC)
|
| Treatment protocol |
For shRNA mediated knockdown, A375 cells were transduced with lentiviruses (LKO.1) that expressed non-targeting control (shSCR or shLuc) or shRNAs specific for TRIM28 (shT28-1 or shT28-2). After selection in puromycin (1 mg/ml), knockdown efficiency was validated by quantitative RT-PCR and immunoblotting.
|
| Growth protocol |
A375 cells were routinely tested for mycoplasma contamination. A375 cells were cultured in high-glucose DMEM (Sigma Aldrich) supplemented with fetal calf serum (10%), streptomycin (0.1 mg/ml), penicillin (100 U/ml), Sodium pyruvate (1 mM) (Sigma Aldrich), HEPES (10 mM) (Sigma Aldrich) and L-glutamine (2 mM) (Sigma Aldrich).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using RNeasy Mini Kit according to the recommended protocol (Qiagen).
|
| Label |
Biotin
|
| Label protocol |
Samples were enzymatically fragmented and biotinylated using the WT Terminal Labeling Kit (Affymetrix).
|
| |
|
| Hybridization protocol |
Hybridization was performed using the Affymetrix hybridization kit. Heat cocktails at 99° for 5 minutes, followed by 42° for 5 minutes. Centrifuge at max speed for 1 minute. Transfer 200μl of hybridization solution to each array. Hybridize 16 hours at 45° at 60rpm. The fluidics protocol was FS450_0001.
|
| Scan protocol |
HTA 2.0 chips were scanned using a GeneChip Scanner 3000 7G.
|
| Description |
shLUC_1
Gene expression data from A375 cells transduced with a non-targeting shRNA.
|
| Data processing |
CEL files were pre-processed using the R package oligo using the robust multichip average (RMA) preprocessing methodology.
|
| |
|
| Submission date |
Jun 20, 2019 |
| Last update date |
Jun 21, 2019 |
| Contact name |
Alexander Espinosa |
| Organization name |
Karolinska Institutet
|
| Department |
Dept of Medicine
|
| Street address |
Visionsgatan 18
|
| City |
Stockholm |
| ZIP/Postal code |
SE-17164 |
| Country |
Sweden |
| |
|
| Platform ID |
GPL17586 |
| Series (1) |
| GSE133073 |
Transcriptome data after TRIM28 knockdown in A375 melanoma cells |
|
