Miseq 150 nt

For TCR library preparation, we used the commercially available iRepertoire platform (iRepertoire, Huntsville, AL, USA) for nested amplicon arm-PCR of the CDR3 of the mouse TCR β-chains and addition of adaptors for Illumina platform sequencing. Reverse transcription of 300–500 ng of RNA was conducted with a One-Step reverse transcription and amplification kit (Qiagen, Germantown, MD, USA) according to the manufacturer’s protocol. The PCR product was purified using Ampure XP magnetic beads (Agencourt/Beckman Coulter, Brea, CA, USA), and secondary amplification of the resulting product was performed (GoTaq PCR Kit, Promega, Madison, WI), allowing addition of Illumina adapter sequences (manufacturer’s protocol). Libraries were purified with Ampure XP magnetic beads and sequenced using Illumina MiSeq 150 nt paired-end read-length. The TCR CDR3 sequences were extracted from the raw sequencing data by iRepertoire.