title: Single-cell RNAseq and scTCRseq of human CD8 T cells from blood and skin compartments in Dengue patients
design: Single-cell RNAseq and scTCRseq of human CD8 T cells from blood and skin compartments in Dengue patients
library_construction_protocol: Whole blood was collected in K3EDTA tubes. PBMCs were isolated from whole blood by Ficoll-Paque gradient centrifugation.  Blisters were induced on the forearm of the patients using a clinical pump connected to a chamber. A negative pressure was applied until a blister was formed and the blister fluid was aspirated using a sterile needle. Skin cells were recovered by centrifugation at 3000 rpm for 10 minutes at 4°C.  CD8+ CD38+ HLA-DR+ cells from matched PBMCs and skin blister cells of dengue patients were sorted into AIM-V + 2% Human Serum on the BD FACSAria™ III Sorter following staining with antibodies targeting surface markers  RNA was extracted from skin blister fluids using the Pathogen 200 protocol on the Roche MagNA Pure 24 Instrument according to the manufacturer's instructions.  Sorted cells were mixed with 10x barcoded gel beads, loaded onto Chromium Next GEM Chip K and ran on the Chromium Controller X to generate GEMs. Post GEM-RT cleanup was then carried out, followed by cDNA amplification according to manufacturer's protocol.  V(D)J amplification from cDNA, V(D)J, library construction as well as 5' gene expression library construction were carried out according to manufacturer's protocol.  Quality control was performed on the Agilent 2100 Bioanalyzer G2938C