|
| Status |
Public on Nov 15, 2023 |
| Title |
shRNA2, biological replicate 5 |
| Sample type |
RNA |
| |
|
| Source name |
769-P cells transduced with shRNA silencing pLKO.1-puro vector
|
| Organism |
Homo sapiens |
| Characteristics |
phenotype: Absence (reduction) of endogenous STAT3
|
| Treatment protocol |
Cells were treated with 10 ng/ml IL6 for 48 h and then RNA was isolated as indicated below
|
| Growth protocol |
Human-derived ccRCC cell lines 769-P (CRL-1933), as well as lentiviruses producer HEK293T/17 (CRL-11268) cell line were cultured in Dulbecco’s Modified Eagle Medium medium supplemented with 10% fetal bovine serum (FBS), 1% sodium pyruvate [100 mM], 1% antibiotic/antimycotic, and 0.2% plasmocin at 37°C with 5% CO2. Culture medium was also supplemented with antibiotics puromycin and blasticidin to select and maintain cell lines transduced with silencing and overexpression vectors, respectively.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using TRIzol reagent, following the supplier’s instructions
|
| Label |
biotin
|
| Label protocol |
Biotinylated ss-cDNA were prepared according to the standard Affymetrix (ThermoFisher) protocol from 200 ng total RNA (GeneChip WT Plus Kit Manual Workflow, user guide).
|
| |
|
| Hybridization protocol |
Following fragmentation, 5,5ug of ss-cDNA were hybridized for 16 hr at 45C on GeneChip Clariom S human array plate. Array plate was washed and stained in the Affymetrix GeneTitan MultiChannel System
|
| Scan protocol |
Array plate was scanned using the Gene Titan MultiChannel System
|
| Description |
Gene expression data from cells after silencing of STAT3
|
| Data processing |
Raw expression values were obtained directly from .CEL files and were processed using the RMA method in oligo R package to get normalized expression values (log2 scale) summarized at the gene level. Different quality checks (boxplots, hierarchical clustering and principal component analyses (PCAs)) were performed before and after normalization to ensure that samples were suitable for differential expression analysis. A complete quality report was obtained using arrayQualityMetrics R package. The analysis to select differentially expressed genes was based on adjusting a linear model with empirical Bayes moderation of the variance. Because batch effects were detected, a batch factor was included in the linear model used for differential expression analysis. P-values are adjusted to obtain strong control over the false discovery rate using the Benjamini and Hochberg method. Statistical language R (version 3.6.0) and Bioconductor associated packages were used to process and analyze the data.
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| |
|
| Submission date |
Oct 20, 2023 |
| Last update date |
Nov 15, 2023 |
| Contact name |
Anna Meseguer |
| E-mail(s) |
ana.meseguer@vhir.org
|
| Organization name |
VHIR
|
| Street address |
Paseo Vall d'Hebron 119-129
|
| City |
Barcelona |
| ZIP/Postal code |
08035 |
| Country |
Spain |
| |
|
| Platform ID |
GPL24324 |
| Series (1) |
| GSE245862 |
STAT3 phosphorylation at serine 727 activates specific genetic programs and promotes clear cell renal cell carcinoma (ccRCC) aggressiveness |
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