GSM6833303: 5_041_01; Homo sapiens; RNA-Seq
PBMCs were isolated using SepMate tubes (Stemcell Technologies) according to the manufacturer's instructions. Briefly, whole blood was diluted 1:1 with phosphate-buffered saline (PBS) and added to a SepMate tube, which contained 15 ml of Ficoll. Tubes were then centrifuged for 10 minutes at 1,200g, after which the PBMC layer was poured off into a fresh tube and washed with PBS. Tubes were then centrifuged at 250 x g for 10 minutes and resuspended in freezing media. Cryovials containing PBMCs were then placed in a CoolCell at -80 º C for 24 hours prior to being transferred to liquid nitrogen for storage. Each sample containing ~1 ml of cryopreserved PBMCs was quickly thawed in a water bath at 37 ºC in medium containing 10% DMSO. 9 ml of warm medium were added and cells were spun at 300 g for 8 minutes. The supernatant was discarded, and 2 ml of medium were added. The samples were filtered with 35 µm cell strainers to obtain a pure single-cell suspension. The samples were mixed with magnetic microbeads to label cell debris, dead cells, and dying cells (Miltenyi Biotec), and the mix was set on a magnet to elute only living cells. The flow through was spun, the supernatant was removed, and the cell pellet was resuspended in 150 µl cell resining buffer with 0.5% BSA (AutoMACS). To enrich for rare cell populations, the samples were split into 2 aliquots: a 30 µl aliquot was kept intact, and a second aliquot was labeled with anti-CD3- Phycoerythrin (PE) antibody (BioLegend) and mixed with anti-PE magnetic microbeads to reduce the number of T cells. Cell numbers in both aliquots were measured, and the two aliquots were remixed. The samples were then diluted to 1000~1400 cells/µl and processed via viscRNA-seq 2.0. The master mix containing 10,000-14,000 cells per sample was prepared following the standard 10X Genomics 5' RNA-Seq protocol. A DENV capturing oligo was added to the master mix at a final concentration of 30 nM for capturing the specific DENV serotype identified via RT-qPCR in patient's sera (DENV1: 5'-﻿AAGCAGTGGTATCAACGCAGAGTACTTTCCCCAGCTTTTCCATGA-3'); DENV3: 5'-﻿AAGCAGTGGTATCAACGCAGAGTACTTTCCCCACGTTTTCCATGA-3'). Samples were loaded into the droplet generator for partitioning single cells into an inverted emulsion. The Next GEM Single Cell 5' reagent kit (10x Genomics) was used for reverse transcription, cDNA amplification and construction of gene expression libraries. ﻿Library quality was assessed using a 2100 Bioanalyzer High Sensitivity DNA kit (Agilent). Sequencing was performed in a paired-end mode with S1 and S4 flow cells ﻿(2 X 150 cycles) on a NovaSeq 6000 sequencer (Illumina).