|
| Status |
Public on Aug 26, 2013 |
| Title |
5nM actinomycin D 2 |
| Sample type |
RNA |
| |
|
| Source name |
A673 Ewing sarcoma cells
|
| Organism |
Homo sapiens |
| Characteristics |
source material: EWS-FLI1-rearranged cell line
cell line: A673
treatment: 5 nM actinomycin D
|
| Treatment protocol |
For knock down experiments, A673 cells were grown to 30-35% confluence in 100 mm dishes at which point growth medium was removed and 5 ml fresh medium added along with 5 ml of virus and Polybrene at a final concentration of 8 μg/ml. After 24 hours, medium was replaced with 2 ml fresh growth medium containing puromycin at 4 μg/ml. Cells were selected for 48 hours, then harvested for Western-blot and RNA extraction.
For drug treatment experiments, A673 cells were grown to 65% confluence and then were either left untreated or treated for 24hrs with DMS0 or actinomycin D at low- (5nM) or high-dose (50nM).
|
| Growth protocol |
A673 cells were obtained from American Type Culture Collection (ATCC) and cultured in DMEM with 10% FCS and RPMI-1640 20 with 15% FCS.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted with Agilent's Absolutely RNA kit.
|
| Label |
biotin
|
| Label protocol |
100 ng of total RNA was submitted to the DFCI Microarray Core Facility for biotin labelling and hybridization to Affymetrix HuGene 1.0 ST Arrays.
|
| |
|
| Hybridization protocol |
Standard Affymetrix protocol.
|
| Scan protocol |
Standard Affymetrix protocol.
|
| Description |
5 nM-24 hrs-2
|
| Data processing |
Array quality was verified before background correction, RMA normalization and log2 conversion using the Bioconductor R package affy (URL: http://www.bioconductor.org).
|
| |
|
| Submission date |
Mar 21, 2013 |
| Last update date |
Aug 27, 2013 |
| Contact name |
Madeleine E. Lemieux |
| E-mail(s) |
mlemieux@bioinfo.ca
|
| Phone |
617-595-6695
|
| URL |
http://www.bioinfo.ca
|
| Organization name |
Bioinfo
|
| Street address |
275 Main St., Suite 252
|
| City |
Plantagenet |
| State/province |
ON |
| ZIP/Postal code |
K0B 1L0 |
| Country |
Canada |
| |
|
| Platform ID |
GPL6244 |
| Series (1) |
| GSE45414 |
Low-dose actinomycin D preferentially disrupts EWS-FLI1DNA binding. |
|
