|
| Status |
Public on Oct 01, 2012 |
| Title |
WZR10 |
| Sample type |
genomic |
| |
|
| Source name |
PC9 NSCLC cells, WZ4002 resistant
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: PC9
wz4002 resistant: yes
|
| Growth protocol |
Cells were grown in RPMI supplemented with 5% FBS. Cells were growing exponentially before the harvest.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was isolated using the DNeasy Blood-Tissue kit (Qiagen).
|
| Label |
biotin
|
| Label protocol |
Labelling was done at the Dana-Farber Cancer Institute (DFCI) molecular biology core. Briefly, the Mapping 250K analysis begins with one aliquot of 250 ng of DNA and is cleaved with StyI. Linkers are ligated to the restriction fragments using T4 DNA ligase. The linkers provide a primer site for the subsequent PCR reaction. The fragments are amplified using Taq polymerase. The PCR products are purified from the primers and free nucleotides by ultrafiltration. They are then quantified spectrophotometrically, and are also assayed using size fractionation on a microfluidics device to determine whether the size distribution of products is as expected. The purified PCR products are fragmented by DNase I in order to provide 3' hydroxyl groups for subsequent labeling, and to facilitate the subsequent hybridization step. The fragmented PCR products are labeled with a single biotin at each free 3'OH using terminal deoxynucleotidyl transferase and a dideoxy biotinylated nucleoside triphosphate.
|
| |
|
| Hybridization protocol |
Hybridization was done at the Dana-Farber Cancer Institute (DFCI) molecular biology core. Briefly, the biotinylated fragments are added to a hybridization solution containing a biotinylated control oligonucletide (for quality control), and hybridized to a microarray chip overnight at 49°C. The chips are then transferred to a fluidics instrument that performs washes to remove DNA that has not hybridized to its complementary oligonucleotide probe. The bound DNA is then fluorescently labeled using phycoerythrin-conjugated streptavidin (SAPE), followed by biotinylated anti-streptavidin, followed by SAPE.
|
| Scan protocol |
Scanning of the microarray chips was done at the Dana-Farber Cancer Institute (DFCI) molecular biology core. Briefly, each DNA bound at its complementary oligonucleotide is excited using a confocal laser scanner, and the positions and intensities of the fluorescent emissions are captured. These measures provide the basis of subsequent biostatistical analysis.
|
| Description |
PC9 WZR10
GR4 cells made resistant to WZ4002, Clone10.
|
| Data processing |
The scanned data was analyzed using D-Chip. The CHP files are the original files from the DFCI core.
|
| |
|
| Submission date |
May 01, 2012 |
| Last update date |
Oct 01, 2012 |
| Contact name |
Pasi Janne |
| E-mail(s) |
pasi_janne@dfci.harvard.edu
|
| Organization name |
Dana-Farber Cancer Institute
|
| Department |
Medical Oncology
|
| Street address |
44 Binney Street Dana 820
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL3720 |
| Series (2) |
| GSE37698 |
Reactivation of ERK signaling causes resistance to EGFR kinase inhibitors (SNP array) |
| GSE37700 |
Reactivation of ERK signaling causes resistance to EGFR kinase inhibitors |
|
