High-Throughput TCR Sequencing Protocol

mRNA was extracted using the Dynabeads mRNA DIRECT purification kit according to the manufacturer instructions (ThermoFisher, cat# 61012). First strand cDNA was synthesized using oligo dT and the Superscript III (Thermofisher). Second strand was performed using a collection of TRAV/TRBV specific primer (1 cycle with the Phusion from NEB). TCRs were then amplified by PCR (20 cycles with the Phusion from NEB) with a single primer pair binding to the constant region and the adapter linked to the TRAV/TRBV primers added during the reverse transcription. A second round of PCR (25 cycles with the Phusion from NEB) was performed to add the Illumina adapters containing the different indexes. The TCR products were purified with AMPure XP beads (Beckman Coulter), quantified and loaded on the MiniSeq instrument (Illumina) for deep sequencing of the TCRα/TCRβ chain. The TCR sequences were further processed using ad hoc Perl scripts to: (i) pool all TCR sequences coding for the same protein sequence; (ii) filter out all out-frame sequences; (iii) determine the abundance of each distinct TCR sequence. TCR with a single read were not considered for the analysis. This methodology was previously reported in Schmidt et al.^{29 (link)}.