Cells grown in RPMI 1640 with 5% FBS (Bio Whittaker, not heat inactivated) and 1% L-glutamine. Cells not grown past 80% confluencey for attached cells, or 0.5 x 10^6 cells for suspended. Trypsinize (for attached cells) cells with 5 ml trypsin-EDTA per T162, for 15 min., at 37C. Spin down suspended cells, and resuspend in ~3ml growth media. Count cells. Pass 1x10^6 cells into new T162's w 30 ml media. Repeat growth cycle until desired amount of cells are available. Cells are not grown past passage 20.
Extracted molecule
genomic DNA
Extraction protocol
Genomic DNA was purified from cells using either the QIAamp DNA Blood Maxi Kit, or the Blood & Cell Culture DNA Maxi Kit (Qiagen Inc., Valencia, CA) according to manufacturer's instructions. Quality was assessed by optical density 260/280 ratio using a spectrophotometer (Beckman-Coulter, Fullerton, CA) and by 0.8% agarose (SeaKem GTG, FMC BioProducts, Rockland, ME) gel electrophoresis in 1x TAE (Roche, Indianapolis, IN).
Label
Cy5
Label protocol
Done according to array manufacturer's specifications.
Cells grown in RPMI 1640 with 5% FBS (Bio Whittaker, not heat inactivated) and 1% L-glutamine. Cells not grown past 80% confluencey for attached cells, or 0.5 x 10^6 cells for suspended. Trypsinize (for attached cells) cells with 5 ml trypsin-EDTA per T162, for 15 min., at 37C. Spin down suspended cells, and resuspend in ~3ml growth media. Count cells. Pass 1x10^6 cells into new T162's w 30 ml media. Repeat growth cycle until desired amount of cells are available. Cells are not grown past passage 20.
Extracted molecule
genomic DNA
Extraction protocol
Genomic DNA was purified from cells using either the QIAamp DNA Blood Maxi Kit, or the Blood & Cell Culture DNA Maxi Kit (Qiagen Inc., Valencia, CA) according to manufacturer's instructions. Quality was assessed by optical density 260/280 ratio using a spectrophotometer (Beckman-Coulter, Fullerton, CA) and by 0.8% agarose (SeaKem GTG, FMC BioProducts, Rockland, ME) gel electrophoresis in 1x TAE (Roche, Indianapolis, IN).
Label
Cy3
Label protocol
Done according to array manufacturer's specifications.
Hybridization protocol
Done according to array manufacturer's specifications
Scan protocol
Done according to array manufacturer's specifications
Description
ME:SK_MEL_2 on Cy5 and Reference on Cy3
Data processing
Normalized data consists of log2 transformed sample/reference intensities. Raw data are the scan files from the Agilent scanner. Log2 ratio of red/green (for arrays where sample=Cy5 and reference=Cy3) or green/red (where sample=Cy3 and reference=Cy5).
