TCR Repertoire Analysis by NGS

The next generation sequencing technology-based TCR repertoire analysis was done as previously described (16 (link),17 (link)). Briefly, total RNA was extracted from tumor sample using RNeasy mini kit (Qiagen, Valencia, CA, USA), and cDNA was synthesized by SMART (Switching Mechanism At 5′ end of RNA Transcript) cDNA library construction kit (Clontech, Mountain View, CA, USA). PCR reactions were performed to amplify compatible amplicon libraries of TCRβ with the Ion Torrent sequencing platform (Life Technologies, Carlsbad, CA, USA). A forward primer (5′-CCTCTCTATGGGCAGTCGGTGATTATCAACGCAGAGTGGCCAT-3′) was designed for the sequence of the SMART IV adaptor and the P1 adaptor. A reverse primer (5′-CCATCTCATCCCTGCGTGTCTCCGACTCAGCAGAAGGAACGATTCTGATGGCTCAAACACAGC-3′) was designed for the constant region of TCRβ, including the A1 adaptor sequence. Underline indicates the IonXpress barcode sequences. PCR reaction was performed as follows: 3 min at 94°C; 40 cycles of 30 sec at 94°C, 30 sec at 65°C, and 35 sec at 68°C. Amplified PCR products were purified using AMPure XP reagent (Beckman Coulter, Brea, CA, USA) and products of 300–900 bp were selected by Pippin Prep system (Sage Science, Beverly, MA, USA). Finally, we measured the concentration of size-selected PCR products by Agilent 2200 TapeStation System (Agilent, Santa Clara, CA, USA).