Purified T lymphocytes (1x106/ml) were stimulated with anti-CD3/anti-CD28 beads (Dynal) at a 1:1 ratio. At six hours, an aliquot of harvested cells was stained with PE-labeled anti-CD4 and FITC-labeled anti-CD69 (BD Pharmingen) to monitor activation.
Growth protocol
Freshly isolated spleens and lymph nodes were disaggregated into DMEM 10% FBS supplemented with L-glutamine, penicillin, streptomycin, sodium pyruvate, HEPES pH 7.4, non-essential amino acids, and β-mercapto-ethanol. Following hypotonic lysis of erythrocytes, cell suspensions were incubated with 1:100 dilutions of biotinylated anti-CD8α, anti-CD11b, anti-CD19, anti-CD49b, anti-TER119, anti-GR-1, and anti-γδ (all BD Pharmingen). Cells were washed once, incubated with anti-biotin magnetic microbeads (1:10, Miltenyi Biotec), washed once more, and purified on magnetic columns. The eluate was stained with PE-labeled anti-CD4 and Cy-Chrome-labeled streptavidin (BD Pharmingen) and examined via flow cytometry to assess purity.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from resting and activated T-cells using RNEasy columns (Qiagen). The quality of the total RNA was verified using a BioAnalyzer (Agilent).
Label
biotin
Label protocol
Sample preparation, labeling and hybridization to Mouse Exon 1.0 ST Arrays (Affymetrix, CA) were performed according to manufacturer’s protocols by the Microarray Core Facility at the Dana Farber Cancer Institute.
Hybridization protocol
Sample preparation, labeling and hybridization to Mouse Exon 1.0 ST Arrays (Affymetrix, CA) were performed according to manufacturer’s protocols by the Microarray Core Facility at the Dana Farber Cancer Institute.
Scan protocol
Sample preparation, labeling and hybridization to Mouse Exon 1.0 ST Arrays (Affymetrix, CA) were performed according to manufacturer’s protocols by the Microarray Core Facility at the Dana Farber Cancer Institute.
Description
transcript (gene)-level data
Data processing
The raw intensity containing CEL files were processed using Affymetrix Power Tools (APT) (http://www.affymetrix.com/support/developer/powertools/index.affx). RMA was performed using updated probe annotations (custom CDFs, Version 8) mapping to Refseq transcripts.
