GSM5901195: 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. The master mix containing 10000~14000 cells per sample were prepared followed 10X protocol. To captured DENV RNA, additional 30nM of customized DENV captured oligos ()were added into the master mix before loading to the ChromiumTM Controller for partitioning single cells into nanoliter-scale Gel Bead-In-Emulsions (GEMs). ﻿The Next GEM Single Cell 5' reagent kit was used for reverse transcription, cDNA amplification and library construction of the gene expression libraries (10x Genomics) following the detailed protocol provided by 10x Genomics. ﻿Libraries quality were checked using 2100 Bioanalyzer with High Sensitivity DNA kit (Agilent). ﻿Sequencing was performed in paired-end mode with a S1 and S4 flow cell ﻿(2 X 50 cycles) using NovaSeq 6000 sequencer (Illumina)