TCR-seq and assessment of the TCR repertoire
Multiplex PCR was performed to amplify the CDR3 regions of the rearranged TCR-β chain from genomic DNA. Thirty-two V-gene specific primers and 13 J-gene primers were used for multiplex PCR. The amplified products were cyclized into single-strand DNA libraries using an MGIeasy Circulation Kit, and then sequenced on the MGI2000 sequencer with 100 bp paired-end reads. VDJtools (v.1.2.1) was used for sequence alignment. TCR repertoire diversity was assessed by productive clonality, which was a measure of species diversity. CDR3 amino acid sequences that had stop or frameshift code, length <5 bp, or did start with ‘C’ or end with ‘F/W’ were considered nonproductive and excluded from subsequent clonotype analyses.TCR richness was defined as the total number of unique clonotypes. A clonality value of 0 represented the most diverse repertoire (each T cell had a unique T cell clonotype), whereas a value of 1 represented a monoclonal T cell population.