BEAM-T GEX library (BEAMT2)
Single-cell TCR sequencing [paired V(D)J, gene expression, and DNA-barcoded multimers] was performed using the Barcode-Enabled Antigen Mapping (BEAM-T, 10X Genomics) pipeline. Briefly, ncHLAp-reactive CTL lines were pooled in equi-cellular ratios and stained with a panel consisting of a viability dye (7-AAD), anti-CD8 (clone SK1, FITC), and the BEAM-T assembly, consisting of a PE-conjugated and molecularly-barcoded peptide-loaded HLA multimers. Staining procedures were performed according to the manufacturer's instructions with minor modifications. Individual BEAM-T assemblies were prepared fresh for each peptide of interest, as well as a negative control assembly. Each candidate BEAM-T assembly was confirmed to have absent background staining prior to incorporation into the panel. Live, CD8+, Tetramer+ singlets were sorted using low-pressure purity mode into an FBS-coated microcentrifuge tube containing 5% HI-FBS. Cells were washed, counted with a fluorescent cell counter, and concentrated to 1000 cells per microliter in PBS for a targeted cell recovery of 10,000 cells. Gene expression, V(D)J and BEAM single-cell libraries were prepared following manufacturer instructions. Library concentrations were quantified with the Agilent Bioanalyzer High Sensitivity assay throughout library construction steps, and final library concentration was determined with the Qubit dsDNA HS Assay kit. Paired-end, dual-index sequencing was performed with a NovaSeq S1 (Illumina) at 10,000 read-pairs per cell, using the 10X recommended sequencing structure. Please refer to 10X Genomics (CG000591_ChromiumNextGEMSingleCell5'v2_BEAM_UserGuide_Rev_B.pdf) for more information.