cd8 t cells: TCR GAG transgenic CD8 cd8 t cell subsets: Memory
After FACS-sorting , T-cells were washed twice with cold PBS and cell pellets were frozen and stored at -80C until all samples were collected. RNA was isolated using Qiagen RNeasy Plus Mini Kit per the manufacturer’s instructions and the yield was determined on a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE). Samples that had an appropriate yield (typically 100ng total RNA) were subsequently analyzed for RNA integrity using an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA).
BeadChips were scanned on an Illumina BeadArray Reader
Memory T cells were generated by transfer of 1x10^6 naïve, transgenic TCR GAG CD8 T cells into B6 mice, followed by i.p. infection one day later with 3x10^7 cfu GAG-expressing Listeria monocytogenes . Splenic CD44hi, CD62Lhi memory TCRGAG T cells were sorted at least two months post infection.
The data was quantile normalized using the Bioconductor package, “lumi,” and transformed to log2 scale