Single read 250bp run

Naive CD4⁺ (CD4⁺CD25^–Foxp3^– ), Triple^loT_reg (CD4⁺CD25^loFoxp3⁺GITR^lo PD-1^lo) or Triple^hiT_reg (CD4⁺CD25^hiFoxp3⁺GITR^hiPD-1^hi) cell populations were sorted from 3 replicate groups (2 mice per group) of single TCRβ-tg (B6.YAe62 $\tilde{\beta }$ tg⁺TCRα^+/–) mice were sorted to 98% purity (FACS Aria, BD Biosciences). RNA was isolated using Trizol and precipitated with RNase free glycogen (Invitrogen) following the manufactures protocol. cDNA was prepared using oligo-dT’s (Promega) and Omniscript RT kit (Qiagen). cDNA was amplified with 20 rounds PCR with generic V_α2 primer (5’-CCCTGGGGAAGGCCCTGCTCTCCTGATA-3’) and TCR Cα primer (5’-GGTACACAGCAGGTTCTGGGTTCTGGATG-3’). 1/10^th volume of the first round PCR was amplified with an additional 20 rounds of PCR using barcoded primers, for post sequence identification of originating T cell population, containing Illunima PE read primer and P5/7 regions, respectively. The resulting 300bp fragment was gel purified (Gene Clean II, MP Biomedicals) and sequenced on a MiSeq using a single read 250bp run (Illumina). Sequence data sets were parsed by barcode using the program fastq-multx59 and clonotypes for each population were tabulated using TCRklass60 (link).