|
| Status |
Public on Oct 13, 2010 |
| Title |
GIST48 ETV1sh1 Rep1 |
| Sample type |
RNA |
| |
|
| Source name |
GIST48 Cell line
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Gastrointestinal stromal tumor cells
cell line: GIST48
shrna: ETV1sh1
replicate: Rep1
|
| Treatment protocol |
Two GIST cell lines (GIST48 and GIST882) were infected in duplicate with three shRNA viruses (Scrambled, ETV1sh1-B11TRCN0000013923, ETV1sh2-TRCN0000013925). Four days after infection, RNA was harvested for expression profiling.
|
| Growth protocol |
GIST48 cells are grown in F10 media supplemented with MITO+, bovine pituitary extract and 15% serum. GIST882 cells are grown in RPMI with 15% serum.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA is extracted using RNeasy per protocol. RNA quality was assessed using the Agilent 2100 Bioanalyzer and the RNA 6000 Nano kit (Agilent Technologies Inc., Palo Alto, CA).
|
| Label |
biotin
|
| Label protocol |
~500 ng of total RNA was used to prepare biotin-labeled RNA using Ambion Illumina TotalPrep RNA Amplification Kit (Cat# AMIL1791, Applied Biosystems, Foster City, CA). Briefly, 500 ng of total RNA was used to synthesize the first strand of cDNA using ArrayScript reverse transcriptase and an oligo(dT) primer bearing a T7 promoter. The single-stranded cDNA was then converted into a double-stranded DNA (dsDNA) by DNA polymerase I in the presence of /E. coli/ RNase H and DNA ligase. After column purification, the dsDNA was served as a template for in vitro transcription in a reaction containing biotin-labeled UTP, unlabeled NTPs and T7 RNA Polymerase.
|
| |
|
| Hybridization protocol |
The amplified, biotin-labeled antisense RNA (aRNA) was purified and quality was assessed using the Agilent 2100 Bioanalyzer and the RNA 6000 Nano kit. 750 ng of aRNA in 5 ul was mixed with 10 ul of hybridization reagents and heated at 65C for 10min. After cooled to room temperature, total 15 ul of the hyb solution was applied to Illumina HumanHT-12 v3 chip. The chip was incubated for about 18 hours at 58C.
|
| Scan protocol |
After washing and staining with streptavidin-Cy3, the chip was scanned using Illumina BeadArray Reader. The scanning was done using standard DirectHyb Gene Expression protocol with the following settings: Factor=1, PMT=587, Filter=100%.
|
| Description |
no additional information
|
| Data processing |
The raw data was extracted using Illumina BeadStudio software without normalization. Raw data was imported into Genespring 10.0.2 for normalization. Detection P-value >0.8 is called present and <0.6 is called absent. Raw signal is threhold to 1.0. The signal is log2 transformed and quartile normalized between samples.
|
| |
|
| Submission date |
Dec 09, 2009 |
| Last update date |
Oct 13, 2010 |
| Contact name |
Yu Chen |
| E-mail(s) |
cheny1@mskcc.org
|
| Phone |
646-888-3356
|
| Organization name |
Memorial Sloan Kettering Cancer Center
|
| Department |
Human Oncology and Pathogenesis Program
|
| Lab |
Chen
|
| Street address |
1275 York Ave, Box 20
|
| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10065 |
| Country |
USA |
| |
|
| Platform ID |
GPL6947 |
| Series (2) |
| GSE19396 |
ETV1 knockdown in GIST cell lines |
| GSE22852 |
Gastrointestinal stromal tumor GIST: gene expresssion and ChIP analyses |
|
