Chromium single cell 5 d j kit

Single T cells were sorted and captured as described above in the method for scRNA-Seq sample preparation. Following first strand cDNA synthesis (Takara) and amplification (Roche), we used 1 uL (of total 25 uL/well) of amplified cDNA for single-cell TCR-sequencing and thus bypassing the RT step as reported previously (Han et al., 2014 (link)). Nested PCR was performed with TCRα/β primers carrying multiplexing barcodes that enabled pooled CDR3α/β sequencing in a single Miseq run. Paired sequencing reads were joined, demultiplexed, and mapped to the human TCR references from the international ImMunoGeneTics information system® (IMGT) with custom scripts as reported previously (Han et al., 2014 (link)). Paired CDR3αβ sequences from the resected tumor of patient A6 were derived using the Chromium Single-Cell V(D)J kit from the 10x Genomics according to the protocol from the manufacturer. For advanced/metastatic lung cancer patients treated with anti-PD1 therapy, bulk TCR sequencing was performed on pre- and post- treatment PBMCs with Immunoseq assay (Adaptive Biotechnologies, Seattle, WA). Single-cell TCR sequencing was performed on post-treatment samples sorted for CD38⁺HLA-DR⁺ cells, as described above.