|
| Status |
Public on Jul 01, 2013 |
| Title |
A673_CloneAsp14_Dox_18h_3 |
| Sample type |
RNA |
| |
|
| Source name |
Asp14 Clone of Ewing's sarcoma A673 cell line with doxycyclin inducible EWS-FLI1 knockdown construct
|
| Organism |
Homo sapiens |
| Characteristics |
treatment: 1 ug/ml doxycyline
time: 18 h
cell line: A673
transgene: doxycyclin inducible EWS-FLI1 knockdown
sample type: Asp14 Clone of Ewing's sarcoma A673 cell line with doxycyclin inducible EWS-FLI1 knockdown construct
|
| Treatment protocol |
18 hours after addition of 1 µg/ml Doxycyline
|
| Growth protocol |
DMEM + GlutaMAXTM-I (+ 4,5g/L D-Glucose + Pyruvate) (Gibco/invitrogen 31966) + Penicillin/Streptomycin (1x) (100x Stock PAA) + 50 µg/ml Zeocin (for the shRNA expression vector) (powder Cayla) and 2 µg/ml Blasticidin (for the TR expression vector)(powder Invitrogen)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extrated using the Qiagen RNA extrction kit according to the manufacturer's instructions.
|
| Label |
biotin
|
| Label protocol |
Target labelling and hybridization. For Affymetrix GeneChip analysis, 2.0µg total RNA was processed to generate a biotinylated hybridization target using One Cycle cDNA Synthesis and One Cycle Target Labelling kits from Affymetrix (Affymetrix, Santa Clara, CA). All procedures were performed according to the manufacturer's protocols (Gene Expression Analysis, Technical Manual, Revison #4; Eukaryotic Target Preparation, Revison #3).
|
| |
|
| Hybridization protocol |
Following RNA purification, 20µg of cRNA were fragmented at 95°C using the Affymetrix fragmentation buffer, mixed with 200µl hybridization buffer containing hybridization controls and hybridized to U133-A2 microarrays. The arrays were stained and washed in an Affymetrix fluidic station 450 following the EukGE-ws2v4 protocol
|
| Scan protocol |
Fluorescence signals were recorded by an Affymetrix scanner 3000 and image analysis performed with the GCOS software (version 1.2).
|
| Description |
Asp14 Clone of Ewing's sarcoma A673 cell line with doxycyclin inducible EWS-FLI1 knockdown, timepoint 18 hours, replica 3
|
| Data processing |
The data was analyzed in R statistical environment using Bioconductor packages. Normalization was done using the gcRMA algorithm
|
| |
|
| Submission date |
Feb 25, 2011 |
| Last update date |
Jul 01, 2013 |
| Contact name |
Maximilian Kauer |
| E-mail(s) |
maximilian.kauer@ccri.at
|
| Organization name |
CCRI, St.Anna Children Cancer Research Institute
|
| Department |
Molecular Biology
|
| Lab |
Heinrich Kovar
|
| Street address |
Zimmermannplatz 10
|
| City |
Vienna |
| ZIP/Postal code |
1090 |
| Country |
Austria |
| |
|
| Platform ID |
GPL571 |
| Series (1) |
| GSE27524 |
A functional liaison between E2F and the aberrant ETS oncogene EWS/FLI1 in Ewing's sarcoma |
|
