One step reverse transcription and amplification kit

TCR sequencing was performed using the Mouse TCR beta, Illumina, V-C genes kit from iRepertoire, Huntsville, AL. In brief, 500 ng of RNA was reverse transcribed using a One-Step reverse transcription and amplification kit (Qiagen). The PCR product was purified using Ampure XP magnetic beads (Agencourt), and amplified again (TopTaq PCR Kit, Qiagen), to add adaptor sequences. 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. Briefly, raw paired-end fastq files were first demultiplexed based on barcode and merged reads were mapped using a Smith-Waterman algorithm to germline V, D, J and C reference sequences from the IMGT web site (http://www.imgt.org). To define the CDR3 region, the position of CDR3 boundaries of reference sequences from the IMGT database were migrated onto reads through mapping results and the resulting CDR3 regions were extracted and translated into amino acids. Reading frames not containing a stop codon were filtered and error-corrected using iRepertoire proprietary SMART algorithm.