Gene expression profiling by Affymetrix and NanoString analysis

Total RNA were extracted from three sets of Tim-3⁺ and Tim-3⁻ Treg. Total RNA from one set were amplified using Nugen Pico WTA System V2 kit to generate adequate amounts of cDNA for downstream Affymetrix genechip analysis using Human Transcriptome Array 2.0 by Genomics and Proteomics Core Laboratories of University of Pittsburgh. The differentially expressed genes and fold of changes were obtained using Affymetrix Transcriptome Analysis Console software. Total RNA from all three sets were subjected to pre-amplification steps to generate sufficient amounts of cDNA for downstream nanostring analysis using nCounter® Human Immunology v2 Gene Expression CodeSet profiling 594 immunology-related human genes and 15 internal reference genes by Genomics and Proteomics Core Laboratories of University of Pittsburgh. Differential expression levels was determined by Student’s t-test.