RNA Isolation and TCR Sequencing
PBMCs were separated by density gradient centrifugation. Total RNA was extracted by QIAamp® RNA Blood Mini Kits (QIAGEN, USA) according to the manufacturer’s instructions. TCR α/β libraries for sequencing were constructed with the SMARTer Human TCR a/b Profiling Kit (TAKARA, USA) following the manufacturer’s protocol. First-strand cDNA synthesis from RNA was primed by the TCR dT Primer and used the SMART-Seq v4 Oligonucleotide for template switching at the 5’ end of the transcript. Following reverse transcription, two rounds of PCR were performed in succession to amplify cDNA sequences corresponding to variable regions of TCR-α and TCR-β transcripts and add the sequencing adapter and sample index. The PCR product was then purified with magnetic beads. The libraries were pooled and sequenced on an Illumina MiSeq sequencer (Illumina, USA) with paired-end, 2 x 300 base pair reads. The used TCR gene regions were determined based on the reference sequences from the Immunogenetics (IMGT) database (V 3.1.34, http://www.imgt.org). All TCR gene segments were determined by MiXCR(16).