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).