Single-cell RNA and TCR sequencing with 10X Genomics platform

Single-cell RNA sequencing was performed using the droplet-based 10X Genomics platform according to the manufacturer’s instructions. T cells and monocytes forming complexes and singlets were sorted as described in the cell sorting section. For ATB, we performed three experiment runs containing six samples each. For Dengue, we performed two experimental runs containing seven and eight samples, respectively. For each experiment run, PBMC samples were stained with a distinct hashtag oligonucleotide antibody as described in the flow cytometry section, to determine the sample origin for each cell after sequencing. Following cell sorting, cells were washed with ice-cold PBS, centrifuged for 10 min (600g at 4°C), and gently resuspended in ice-cold PBS supplemented with 0.04% ultrapure bovine serum albumin (Sigma-Aldrich). Cells were sorted by flow cytometry into low retention 1.5 ml collection tubes, containing 500 μl of PBS:FBS (1:1) supplemented with RNase inhibitor (1:100). After sorting, ice-cold PBS was added, cells were spun down, and single-cell libraries were prepared as per the manufacturer’s instructions (10X Genomics). Samples were processed using 10× 5’v2 chemistry as per the manufacturer’s recommendations. The library preparation was performed using Chromium Next GEM Single cell Standard 5V2 (Dual index) with feature Barcode technology kit and chromium Single Cell Human TCR Amplification Kit. Libraries were sequenced using the Illumina NovaSeq 6000 sequencing platform.