GSM8124005: VCOV_s03_P1,γδ scTCR-seq; Homo sapiens; OTHER
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). 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 γδ scTCR-seq libraries. To this end, gene-specific primers 25 were used within the 5′ regions of the TRGC and TRDC segments for the enrichment of TCR transcripts (v1.1 chemistry) Complementary DNAs were amplified, and the quality was assessed using an Agilent 4200 Tapestation. The scTCR-seq libraries were sequenced on Illumina NextSeq 550 platform.