Transcriptome analysis of naïve Tregs, memory Tregs and CD161+ Tregs

We analyze bulk transcriptomes of three populations of human Tregs 200,000 naïve, memory and CD161+ Tregs were either sorted from fresh blood directly into Trizol LS (Ambion) for baseline genetic profile or cells were polyclonally activated post-sorting for 4h with Dynabeads Human T-Activator CD3/CD28 (ratio 1:1) and then lyzed in Trizol LS. RNA was isolated using RNeasy mini kit (QIAGEN) and Ovation PicoSL WTA System V2 (NuGEN Technologies) was used for reverse transcription and cDNA amplification steps. Fragmentation and labelling was performed using the Encore Biotin Module (NuGEN Technologies); all kits were employed following manufacturer’s instructions. Samples were run on GeneChip® Human Gene 1.0 ST Array (Affymetrix Ltd).