GSM8123920: VCOV_s06_P0,scRNA-seq; Homo sapiens; RNA-Seq
Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats of HDs (n=6) or from 6 healthy vaccinated subjects through Lympholyte®-H Cell Separation density gradient solution (Cedarlane Laboratories, Burlington, North Carolina, USA) according to the manufacturer's instruction. In vitro experiments were performed on freshly isolated PBMCs, with a viability greater than 95%, while single-cell ones on PBMCs previously frozen in Fetal Bovine Serum (FBS) supplemented with 10% of the cryoprotective Dimethyl Sulfoxide (DMSO). Libraries for scRNA-seq were prepared using the Chromium Single Cell Platform with a Single-Cell 5′ Library and Gel Bead Kit (10X Genomics, PE-1000006). Thawed PBMC samples were evaluated for viability prior to scRNA-seq analysis, and all were ≥95% viable. Cells were resuspended in a volume equivalent to 10,000 target cells for each sample, and were individually loaded onto a Chromium single-cell controller (10X Genomics) to generate single-cell gel beads-in-emulsion (GEMs). Captured cells were then lysed and the released RNA was barcoded through reverse transcription in individual GEMs. Complementary DNAs (cDNA) were generated and split to generate the additional  γδ scTCR-seq libraries. Complementary DNAs were amplified, and the quality was assessed using an Agilent 4200 Tapestation. The scRNA-seq libraries were sequenced using an Illumina Novaseq6000 sequencer with a paired-end 150-bp (PE150) reading strategy (performed by CapitalBio Technology). scRNA-seq paired with γδTCR-seq