title: single-cell TCR-seq of treatment-naïve HNSCC: Sample p20-C-0024_2_A
design: Fresh pre-treatment tumor biopsies were digested using the Human Tumor Dissociation Kit and the gentleMACS Dissociator (Miltenyi Biotec) per manufacturer recommendations. Single cell tumor suspensions were washed with RPMI-1640, passed through a 70-mm filter, stained with TruStain FcX (Biolegend), primarily-conjugated anti-human CD3 antibody (clone SK7, Biolegend) and the viability marker Sytox Blue (Thermo) and viable CD3-positive cells were isolated using fluorescent activated cell sorting (FACSAria III, BD Biosciences). Cells were washed in 1× PBS, filtered (70 µm) and quantified using acridine orange/propidium iodide (AO/PI) staining on a Cellometer Auto 2000 (Nexcelom). Cells were concentrated to 1000 cells/µL and loaded onto the Chromium Controller (10X Genomics) with a target of 6,000 cells per sample. Cells were mixed with barcoded gelbeads and 5' GEM Kit v1.1 reagents (10X Genomics), and single-cell capture was performed. Following reverse transcription, cDNA was amplified, and a TCR library was constructed according to the manufacturer's recommendations. Each DNA library was loaded into a sequencing lane on a NextSeq system (Illumina) and was sequenced with pair-end reads of 75 bp.