Multiplexed αβ-TCR Sequencing Protocol

Multiplexed αβ-TCR sequencing was done following previously established protocols (33 (link)). In brief, single spheromer⁺ CD8⁺ T cells (for influenza-M1, HCMV-pp65, and SARS-CoV-2 specificities) were sorted into 96-well plates containing 12 μl of OneStep RT-PCR buffer (Qiagen). Reverse transcription was done using the OneStep RT-PCR kit (Qiagen), and the resulting complementary DNA (cDNA) was used for TCRα and TCRβ amplification using multiplex primers. DNA barcodes were also incorporated within the amplified sequences before processing the samples in a single MiSeq2 × 300–base pair sequencing run. The paired sequencing reads were joined, demultiplexed, and mapped to the human TCR reference dataset available at the international ImMunoGeneTics information system as reported previously (33 (link)).