Single cell RNA-Seq of dengue seronegative and seropositive individuals
Blood was collected in EDTA tubes from subjects, and peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation with SepMate-50 tubes (Stemcell Technologies) according to manufacturer's instructions. Frozen PBMCs were thawed and cells were counted using Nexcellom Cellometer Spectrum, and cell viability assessed using Live/DEAD Viability/Cytotoxicity Kit for mammalian cells (Thermo Scientific). Cells are then partitioned into nanoliter-scale gel beads-in-emulsion (GEMs). Gel beads are dissolved, primers are released and any co-partitioned cell is lysed. Incubation of GEMs generates barcoded, full length cDNA from poly-adenylated mRNA.  Single cell library preparation was performed using 10x Genomics Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (Dual Index) by Molecular Genomics Pte Ltd. Profiles of cDNA samples and the final libraries were verified using Agilent High Sensitivity D5000 screentapes on an Agilent Tapestation 4200. At least 5000 cells were targeted per sample with at least 20,000 reads sequenced per cell.