|
| Status |
Public on Sep 24, 2013 |
| Title |
HepG2_cells_5000nM-RVX-208-treatment_4h_replicate_3 |
| Sample type |
RNA |
| |
|
| Source name |
HepG2 cells, RVX-208 treatment (5000nM), 4h
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HepG2
|
| Treatment protocol |
Cells were treated for 4h with (1) DMSO or (2) 500nM JQ1 or (3) 5000nM RVX-208
|
| Growth protocol |
HepG2 cells (ATCC: HB-8065) were maintained in α-MEM (Cat.#BE12-169F; BioWhittaker) supplemented with 10 % heat-inactivated foetal calf serum (PAA #A15-152), non-essential amino acids (Cat. #M7145; Sigma), glutamine (Cat.#M11-004; PAA), and vitamins (Cat.#M6895; Sigma). Cells were grown at 37 °C in a humidified cabinet at 5 % CO2 (Heraeus Function Line). For experiments, cells were seeded the day prior to treatment at 2x105/ml. Treatments were performed for 4 h so that a final concentration of 0.1 % DMSO (Cat.#D1435; Sigma) was achieved. At harvest, cells were washed once with PBS (Cat.#H15-002; PAA), and lysed in situ using RLT buffer supplemented with 10 μl/ml β-mercaptoethanol (Cat.#M7522; Sigma).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted and prepared using RNeasy columns (Cat.#74106 plus; Qiagen) including a Qia shredding step (Cat.#79656; Qiagen) and an on-column DNAse digestion (Cat.#EN0521; Fermentas), according to the manufacturer’s instructions
|
| Label |
biotin
|
| Label protocol |
200ng of total RNA were amplified and labelled using the Ambion WT expression kit and the Affymetrix GeneChip WT Terminal Labelling and Controls Kit
|
| |
|
| Hybridization protocol |
Affymetrix GeneChip® Human Gene 1.0 ST arrays were hybridized for 17h to 11 mg of labeled, amplified cDNA, washed, stained and scanned according to the protocol described in WT Sense Target Labeling Assay Manual
|
| Scan protocol |
Chips were processed on an Affymetrix GeneChip Fluidics Station 450 and Scanner 3000
|
| Description |
Sample name: RVX3
Gene expression data from HepG2 cells treated with 5000nM RVX-208 for 4h
|
| Data processing |
The Affymetrix Command Console (v.3.2.4; Affymetrix) was used to generate CEL files. Raw CEL data were processed in R (v.2.15.3) using Bioconductor (v.2.11) and the simpleaffy package (v.2.34.0). Quality controls were carried out using the arrayQualityMetrics package (v.3.14.0) taking into account array intensity distributions, distance between arrays and variance mean-dependence. Principal component analysis was used to decide which arrays to process together. On this basis one of the three arrays treated with JQ1 was considered to be an outlier and was discarded (sample 'JQ3', file 'JQ3_(HuGene-1_0-st-v1).cel'), with the rest of the analysis performed without taking it into account. Background correction and normalization were carried out employing the Robust Multichip Array (RMA) method implemented in the affyPLM package (v.1.34.0). Data were filtered using the genefilter package (v.1.40.0) and probe sets that did not have an Entrez gene identifier were removed. From the 32321 probe sets available on the Human Gene 1.0 ST chip, 19816 remained after filtering. A linear model was applied employing the limma package (v.3.14.4) followed by empirical Bayesian analysis to determine differential expression between not-treated and treated samples. Data were annotated using the hugene10sttranscriptcluster.db annotation data (http://www.bioconductor.org/packages/2.11/data/annotation/html/hugene10sttranscriptcluster.db.html).
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| |
|
| Submission date |
Sep 24, 2013 |
| Last update date |
Sep 24, 2013 |
| Contact name |
Panagis Filippakopoulos |
| E-mail(s) |
panagis.filippakopoulos@gmail.com
|
| Organization name |
Oxford University
|
| Department |
Nuffield Department of Medicine
|
| Lab |
Structural Genomics Consortium
|
| Street address |
ORCRB - Roosevelt Drive
|
| City |
Oxford |
| State/province |
Oxfordshire |
| ZIP/Postal code |
OX3 7DQ |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL6244 |
| Series (1) |
| GSE51143 |
Effect of BET inhibitors (JQ1 and RVX-208) on gene expression in HepG2 cells |
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