|
| Status |
Public on Nov 22, 2011 |
| Title |
V 12 CMV PBMC |
| Sample type |
RNA |
| |
|
| Source name |
CMV PBMC
|
| Organism |
Homo sapiens |
| Characteristics |
patient code: V 12
disease: melanoma
tissue: peripheral blood
cell type: CD8+ T cells
tcr specificity (or naive): CMV (pp65 495-503)
age: 95 years
Sex: male
|
| Treatment protocol |
Mononuclear cells were purified by density gradient using Lymphoprep and immediately frozen. CD8+ T cells were enriched using magnetic bead sorting. Cells were stained on ice and then diluted at one million cells/ml for optimal sorting speed and high purity.
|
| Growth protocol |
Samples were processed ex vivo.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Using a Vantage SE, 1000 cells from each cell population were sorted directly into lysis and storage buffer, as provided by Miltenyi Biotec. After sorting, the lysed cells were incubated for 10min at 45°C and then directly frozen at –80°C.
|
| Label |
Cy3
|
| Label protocol |
250 ng of each of the cDNAs were used as template for Cy3 labeling, which was performed according to Miltenyi Biotec’s undisclosed protocol.
|
| |
|
| Hybridization protocol |
The Cy3-labeled cDNAs were hybridized overnight (17 hours, 65°C) to an Agilent Whole Human Genome Oligo Microarray 4 x 44K using Agilent’s recommended hybridization chamber and oven. Microarrays were washed once with 6x SSPE buffer containing 0.005% N- lauroylsarcosine for 1 min at room temperature followed by a second wash with pre-heated 0.06x SSPE buffer (37 °C) containing 0.005% N-lauroylsarcosine for 1 min. The last washing step was performed with acetonitrile for 30 sec.
|
| Scan protocol |
Fluorescence signals of the hybridized Agilent Microarrays were detected using Agilent’s Microarray Scanner System (Agilent Technologies).
|
| Description |
Set 2
Gene expression ex vivo of CMV-specific T cells from blood.
|
| Data processing |
Raw intensities from the Feature Extraction files were used and analyzed using R and Bioconductor applying the Agi4x44PreProcess package.
|
| |
|
| Submission date |
Oct 05, 2010 |
| Last update date |
Nov 22, 2011 |
| Contact name |
Lukas Baitsch |
| E-mail(s) |
lukas_baitsch@dfci.harvard.edu
|
| Organization name |
Dana Farber Cancer Institute
|
| Department |
Cancer Biology
|
| Lab |
Jean Zhao
|
| Street address |
450 Brookline Avenue
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02215 |
| Country |
USA |
| |
|
| Platform ID |
GPL6480 |
| Series (1) |
| GSE24536 |
Molecular signature of CD8+ T cell exhaustion in metastases but not in peripheral blood from melanoma patients |
|
