Next-Generation Sequencing of T Cell Receptor Repertoire

CD4⁺CD45RO⁺ and CD8⁺CD45RO⁺ T cells were prepared as described above. The T cells were lysed with Isogen-LS (NIPPON GENE) and stored at − 80 °C. The lysates were sent to Repertoire Genesis Inc. (Ibaraki, Japan) for next-generation sequencing, which was performed as previously described^{51 (link)}. Briefly, total RNA was converted to complementary DNA (cDNA) with the SuperScript reverse transcriptase (Invitrogen). Double-stranded cDNA was synthesized and ligated with a 5′ adaptor oligonucleotide, then cut with the SphI restriction enzyme. Next, the double stranded cDNA was amplified through polymerase chain reaction using primers specific for the adaptor and TCRα constant region. The sequencing was performed with the Illumina MiSeq paired-end platform (2 × 300 bp). Data processing was performed with the repertoire analysis software developed by Repertoire Genesis Inc^{52 (link)}. TCR sequences were assigned with a dataset of reference sequences from the international ImMunoGeneTics information system database (http://www.imgt.org). The percentage of sequence reads with TRAV, TRAJ, TRBV, and TRBJ genes was calculated. The Circos plots were produced using the Circos software package^{53 (link)}. The Shannon–Weaver index shows the diversity and is defined as follows. $$\mathrm{Shannon}-\mathrm{Weaver}\mathrm{index}=-{\sum }_{\mathrm{i}-1}^{\mathrm{S}}\left(\frac{{\mathrm{n}}_{\mathrm{i}}}{\mathrm{N}},\ln ,\frac{{\mathrm{n}}_{\mathrm{i}}}{\mathrm{N}}\right)$$