|
| Status |
Public on Nov 07, 2013 |
| Title |
SK-MEL-28.GFP-sh.rep1 |
| Sample type |
RNA |
| |
|
| Source name |
SK-MEL-28, GFP-sh, 4 days, rep1
|
| Organism |
Homo sapiens |
| Characteristics |
cell line type: melanoma cell line
cell line: SK-MEL-28
treatment: GFP-sh, 4 days
|
| Treatment protocol |
24 hours post-lentiviral infection add 1µg/ml puromycin for 4 days
shRNA expression was performed for 4 days. Coumpound treatment was 20 µM for 16 hours. Final DMSO concentration (treatment and control) was at 0.2%.
|
| Growth protocol |
Cells were grown in RPMI media supplemented with 10% FBS
|
| Extracted molecule |
total RNA |
| Extraction protocol |
QIAgen kit according to manufacturer protocol
Total RNA from the samples was normalized to 50 ng/ul and the GeneChip® 3’ IVT Express Kit (Affymetrix PN 901253) was used for amplification according to the standard protocol. The total RNA underwent reverse transcription to synthesize first-strand cDNA. This cDNA was then converted into a double-stranded DNA template for transcription. In vitro transcription synthesized aRNA and incorporated a biotin-conjugated nucleotide. The aRNA was then purified to remove unincorporated NTPs, salts, enzymes, and inorganic phosphate. Fragmentation of the biotin-labeled aRNA prepared the samples for hybridization to Affymetrix HT-HG U133 A peg arrays (Affymetrix PN 900751).
|
| Label |
biotin
|
| Label protocol |
Labeling was conducted manually, using the Affymetrix 3' IVT Express Kit (Affymetrix PN 901253) according to the attached standard protocol. Reactions were cycled on the Thermo Hybaid MBS Satellite 0.2G cycler. http://www.affymetrix.com/estore/esearch/search.jsp?pd=131549&N=429496729
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| |
|
| Hybridization protocol |
Hybridization and scanning were conducted using the Affymetrix GeneTitan (MC) instrument (Affymetrix PN 00-0372), according to the attached standard protocol.http://www.affymetrix.com/estore/esearch/search.jsp?Ntt=titan&basic=1
|
| Scan protocol |
CEL files were produced using the Affymetrix GeneChip Command Console software (Affymetrix PN agcc01) integrated with the GeneTitan. http://www.affymetrix.com/estore/browse/products.jsp?productId=131429#1_1
|
| Data processing |
The data were analyzed with R 3.0.2, using the package affy_1.40.0, and RMA as normalization method. RMA was used with default options (normalize=TRUE, background=TRUE) and with the package hthgu133ahsentrezgcdf v17.1.0 from Brainarray for the CDF file.
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| |
|
| Submission date |
Nov 06, 2013 |
| Last update date |
Nov 07, 2013 |
| Contact name |
Marius Pop |
| E-mail(s) |
[email protected]
|
| Organization name |
DFCI
|
| Street address |
450 Brookline ave
|
| City |
Boston |
| ZIP/Postal code |
02215 |
| Country |
USA |
| |
|
| Platform ID |
GPL17897 |
| Series (1) |
| GSE52154 |
Gene expression signature overlap between shETV1 and BRD32048 in LNCaP and SK-MEL-28 |
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