GSM5682618: VDJ-seq_EXP20000910_Sample_12; Homo sapiens; RNA-Seq
PBMCs were thawed, and CD8+ T cells were enriched by magnetic-activated cell sorting (MACS) using a CD8+ T Cell Isolation Kit (Miltenyi) following the manufacturers protocol. The CD8+ T cells were then stained with 1 nM final concentration tetramer library in the presence of 2 mg/mL salmon sperm DNA in PBS with 0.5% BSA solution for 20 minutes. Cells were then labeled with anti-TCR antibody-derived tag (ADT, IP26, Biolegend) for 15 minutes followed by washing. Tetramer bound cells were then labeled with phycoerythrin (PE) conjugated anti-DKDDDDK-Flag antibody (BioLegend) followed by dead cell discrimination using 7-amino-actinomycin D (7-AAD). The live, tetramer positive cells were sorted using a Sony MA900 Sorter (Sony). Tetramer positive cells were counted by Nexcelom Cellometer (Lawrence, MA, USA) using AOPI stain following manufacturer's recommended conditions. Single-cell encapsulations were generated utilizing 5' v1 Gem beads from 10x Genomics (Pleasanton, CA, USA) on a 10x Chromium controller and downstream TCR, and Surface marker libraries were made following manufacturer recommended conditions. All libraries were quantified on a BioRad CFX 384 (Hercules, CA, USA) using Kapa Biosystems (Wilmington, MA, USA) library quantified kits and pooled at an equimolar ratio. TCRs, surface markers, and tetramer generated libraries were sequenced on Illumina (San Diego, CA, USA) NextSeq550 instruments. Sequencing data were processed using the Cell Ranger Software Suite (Version 3). Samples were demultiplexed and unique molecular identifier (UMI) counts were quantified for TCRs, tetramers, and gene expression. 10X Chromium 5' single-cell
GEO Accession
GSM5682618