Single-cell Transcriptomic and Immune Receptor Profiling of PBMCs from Dengue-Infected Individuals with Varying Disease Severities
Single-cell Transcriptomic and Immune Receptor Profiling of PBMCs from Dengue-Infected Individuals with Varying Disease Severities
Frozen peripheral blood mononuclear cells (PBMCs) were thawed at 37°C and diluted five times 1:1 in RPMI containing 10% FBS, following the 10x Genomics protocol for fresh frozen human PBMCs. Dead cells were removed using a Dead Cell Removal Kit (Miltenyi Biotec), and cell clumps were filtered with 40 µm Flowmi® strainers (SP Industries). Cell counts were determined microscopically prior to single-cell capture. No separate RNA extraction was performed. Single-cell suspensions were directly loaded into the 10x Chromium™ Controller for gel bead-in-emulsion (GEM) generation and reverse transcription. Reverse transcription, cDNA amplification, and library construction were performed using Chromium™ Single Cell V(D)J Reagent Kits with V1 Chemistry (10x Genomics), following the manufacturer's protocol. Target recovery was 10,000 cells per sample without sample multiplexing.