|
| Status |
Public on Jul 22, 2013 |
| Title |
Cyclophosphamide 5mM, biological rep1 |
| Sample type |
RNA |
| |
|
| Source name |
Jurkat, P16
|
| Organism |
Homo sapiens |
| Characteristics |
passage: P16
cell type: Human T cell
agent: Cyclophosphamide 5mM
cell line: Jurkat, Clone E6-1
|
| Treatment protocol |
Jurkat cells (passage number 16 to 20) were seeded in 2.7 ml medium per well in 6-well plates (750,000 cells/well) . After growing the cells for 20 hours, exposure was initiated by adding 0.3 ml medium containing non-cytotoxic concentration of the compounds or vehicle controls. Subsequently, cells were exposed to the compounds for 6 h. The final DMSO concentration in the medium was 0.1% (v/v) for all the samples. Each exposure was performed in quadruplicate at four different days.with 0,7% NaCl, 0,04% triton-X100 and placed on ice in the Trizol solution (GibcoBRL).
|
| Growth protocol |
The Jurkat cells were grown in RPMI-1640 medium supplemented with 10% heat inactivated Fetal Calf Serum (FCS), 2 mM glutamine, 1 mM sodium pyruvate, 1 mM non-essential amino acids, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were cultured at 37 °C with 5% CO2 in a humidified atmosphere. The medium was refreshed every 2 days.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated by using Qiagen QIAshredder kit according to the manufacturer’s protocol. RNA purification was done using Qiagen miRNeasy Mini kit.
|
| Label |
biotin
|
| Label protocol |
cDNA synthesis and subsequent synthesis of biotin-labeled cRNA was performed using GeneChip One-Cycle Target Labeling and Control Reagents including the One-Cycle cDNA Synthesis Kit, Poly-A RNA Control Kit, Sample Cleanup Module, IVT Labeling Kit, and Hybridization Control Kit (all from Affymetrix) according to the manufacturer’s protocol.
|
| |
|
| Hybridization protocol |
cRNAs were hybridized to the arrays for 16 hours at 45 °C (GeneChip Hybridization Oven 640; Affymetrix). Washing and staining were done by using GeneChip hybridization station, Fluidics Station 450, Affymetrix.
|
| Scan protocol |
GeneChips were scanned by Affymetrix GeneChip Scanner 3000 7G.
|
| Data processing |
Raw data were extracted using GeneChip Operating Software (Affymetrix). In order to filter out probes with sub-optimal specificity for the encoding genes, custom CDF files were generated from the raw data files, by using the R package available at http://brainarray.mbni.med.umich.edu/Brainarray/Database/CustomCDF/14.1.0/entrezg.asp. Then robust multichip average (RMA) normalization was applied to the complete dataset (Bioconductor). BioConductor packages were used for the quality control of the microarray data (www.arrayanalysis.org).
|
| |
|
| Submission date |
May 14, 2013 |
| Last update date |
Jul 22, 2013 |
| Contact name |
Jia Shao |
| E-mail(s) |
jia.shao@wur.nl
|
| Organization name |
RIKILT-Institute of food safety
|
| Department |
TE
|
| Street address |
Akkermaalsbos 2
|
| City |
Wageningen |
| ZIP/Postal code |
6708 WB |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL16311 |
| Series (1) |
| GSE46909 |
Expression data from human Jurkat T cells exposed to 31 compounds |
|
