Rag-/- TCR mice were immunized twice with 5x106 irradiated FBL and 10 µg anti-CD40 (Immunex, Seattle, WA) and 30 days later spleens from immunized Rag-/- TCR and non-immunized Rag-/- TCR and TCRxGag were removed. Naïve CD44low and CD44high memory and tolerant CD8+ T cells were isolated by cell sorting on a FACSAria (Becton Dickinson, San Jose, CA). RNA was extracted from sorted T cells using Trizol® Reagent (Invitrogen, Carlsbad, CA) and then purified further using an RNeasy cleanup kit from Qiagen (Valencia, CA) according to manufacturer’s protocol. Total RNA was quantified using RiboGreen (Molecular Probes, Eugene, Oregon)
Label
biotin
Label protocol
samples were split into equal aliquots of ~50ng of total RNA, and used for three rounds of cDNA synthesis and cRNA amplification, as previously described (Scherer, A. et al. Optimized protocol for linear RNA amplification and application to gene expression profiling of human renal biopsies. Biotechniques 34, 546-50, 552-4, 556 (2003).)
Hybridization protocol
standard Affymetrix procedures except for use of a custom 24-channel fluidics machine
