PBMCs were purified by a standard Ficoll gradient method and cultured in RPMI 1640 medium containing 10% FBS plus antibiotics. Subsequently, PBMCs (2 x 10^6 cells/culture) were suspended in RPMI 1640 medium containing 10% FBS supplemented with 8 ug/ml polybrene, and infected with rKSHV.291 at MOI=1. At various time points (day 0, 1, 2, and 4) after infection, the cells for each group were washed and fixed with paraformaldehyde according to the 10x Genomics Cell Preparation Guide (CG000053). Single-cell suspensions (1x10^4 cells) were processed on the Chromium Controller (10x Genomics) for cell partitioning into individual Gel Bead-In-EMulsions (GEMs), generation of barcoded cDNAs containing containing an Illumina R1 primer sequence and unique molecular identifiers (UMIs), cDNA amplification, and construction of barcoded sequencing libraries using the Chromium Single Cell 3' Reagent Kit v2 (10x Genomics). Prior to library construction, cDNA amplicon size was optimized by enzymatic fragmentation and size selection. Subsequently, Illumina P5 and P7 adaptors, a sample index, and Illumina R1 primer sequence are incorporated during library construction via end repair, A-tailing, adaptor ligation, and PCR.