TCR beta repertoire of T cells from immunized Balb/c
7-week-old mice were immunized subcutaneously with 10μg of HEL (Sigma) or PBS (Gibco) emulsified in CFA (DIFCO).  Draining lymph nodes were harvested on day 9.   Cells were suspended at a concentration of 5 x 106 cells per milliliter in chemically defined, HL-1 serum-free medium (Lonza) supplemented with streptomycin and penicillin (Gibco) and cultured for 3 days with 10 μg /ml HEL, 20ng/ml of PPD, 2 μg /ml of ML2  or 2 μg /ml of ML1 peptide.    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.    High-throughput sequencing was done on an Illumina HiSeq 2000 platform. All amplicon libraries were indexed using Nextera XT Index kit (Illumina Inc.) according to the manufacturer's instructions for  " 16S Metagenomic Sequencing Library Preparation" protocol with minor modifications.   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.
cfa/pbs-med