GSM5065336: MELA31_rep2 T0; Homo sapiens; RNA-Seq
Cryopreserved PBMC were thawed in R10 supplemented with 20 μg/ml DNase I from bovine pancreas (Sigma-Aldrich). After washing with phosphate buffer saline (PBS), cells were stained with the Red Live Dead Fixable Viability kit (ThermoFisher) for 15 min at room temperature. PBMC were washed with PBS and stained with mAb anti-CD3-PE and -CD8-FITC (Biolegend). Viable CD3+, CD8+ T cells were sorted by using eS3 sorter. Cell sorting was performed with 96-99% purity. Cell suspension were then visualized and counted with trypan blue using Bio-Rad T20 Automated Cell Counter to get a precise estimation of total number of sorted cells and of cells concentration. Cells were then pelletted and resuspended in the appropriate volume of cold 1X PBS + 0.1% BSA until reached the final concentration of 3000 cells/μl. Afterwards we loaded about 54.000 cells of each sample into one cartrige of the ddseq system. Following capture and lysis, cDNA was synthesized and amplified for 15 cycles following the Illumina® Bio-Rad® SureCellTM WTA 3′Library Prep for Peripheral Blood Mononuclear Cells (PBMC) protocol. 50 ng of the amplified cDNA were then used for each sample to construct Illumina sequencing libraries. Sequencing was performed on the NextSeq550 Illumina sequencing platform following Illumina® Bio-Rad® instruction for read generation, reaching at least 50000 reads as mean reads per cell.