TCR beta repertoire of CD4 T cells from Balb/c
Total RNA was isolated using the Qiagen RNEasy Plus Kit (Qiagen) and treated with RNA-free DNase I (Ambion) to remove genomic DNA. RNA concentrations were quantified using a Qubit® Fluorometer and the Quant-iT RNA assay kit (Invitrogen). Reverse transcription was performed using the TCR Cβ-specific primer MCβ12RT and the SuperScript® III First-Strand Synthesis System for RT-PCR (Invitrogen) according to the manufacturer's instructions.   Quantitative real-time PCR was performed for each sample to identify the minimum number of PCR cycles needed to ideally amplify TCR beta without over amplification. We carried out qRT-PCR with the primer pairs MVβ8.2) and MJβ1.5 and used the Platinum® Taq DNA Polymerase HiFi kit (Invitrogen) and EvaGreen® Dye (Biotium). Once the optimal number of PCR cycles was established, final PCR was carried out with primer pairs MVB8.2 and MJb1.5, and the Platinum® Taq DNA Polymerase HiFi kit (Invitrogen). All amplification reactions were assayed in at least 10 separate tubes to minimize PCR amplification bias. The reaction products were purified using the MinElute PCR Purification Kit (Qiagen) and quantified using a Qubit® Fluorometer and Quant-iT dsDNA BR Assay Kit (Invitrogen).   High-throughput sequencing was done on an Illumina HiSeq 2000 platform. All libraries were prepared using TruSeq DNA Sample Preparation Kit v2 (Illumina Inc.) according to the manufacturer's instructions for gel method version of TruSeq DNA Sample Prep v2 Low Throughput protocol with minor modifications.   Briefly 300-500 ng of PCR fragment was used for each library.   MinElute PCR Purification Kit (Qiagen) was used to purify DNA after end-repaired step.   E-gel SizeSelect 2% gel (Invitrogen) was used to purify adaptor ligated products. Enriched library were purified using Agencourt® AMPure® Kit (Beckman Coulter, Inc). Enriched library were quantified using a Qubit Fluorometer and Quant-iT HS DNA assay kit (Invitrogen)   Sequencing was performed in 100-bp paired-end mode with Illumina HiSeq 2000 chemistry. Raw sequencing reads were processed for FASTQ conversion and demultiplexing using CASAVA 1.8.2.