NextSeq 500 paired end sequencing; Single cell RNA-seq of HLA-E restricted UL40-specific T cells
Single cell RNA-seq of HLA-E restricted UL40-specific T cells
Blood samples were collected in sodium heparin vacutainer tubes (Becton Dickinson) and peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-plaque (GE Healthcare). PBMC were cryopreserved in 10% DMSO 90% fetal calf serum until analysis. PBMCs were thawed and cultured overnight in RPMI containing 10% human AB serum (Sigma) with 10 U/ml recombinant IL-2. PBMC from the donor TSF were stained with HLA-E/VMAPRTLIL tetramer-PE and CD8β-PECy5. Single tetramer+CD8β+ cells at high or low tetramer staining intensity were sorted directly into individual wells of separate 96-well plates (Eppendorf) using a FACSAria I (Becton Dickinson) at 80 cells per plate into a dNTP mix and lysis buffer. Libraries were prepared with the modified Smart-seq2 protocol essentially as described in (Picelli, S. et al. Full-length RNA-seq from single cells using Smart-seq2. Nat Protoc 9, 171-181 (2014)). Nextera XT DNA sample preparation kit and Index kit A and D (Illumina).