GSM6568490: Hu blood mono, replicate 3, scRNAseq; Homo sapiens; RNA-Seq
Peripheral blood was obtained from 5 adult male healthy donors. Samples were collected in EDTA-coated tubes (BD Vacutainer K2E), and peripheral blood mononuclear cells (PBMCs) were isolated by Lympholyte® Cell Separation density gradient solution (Cederlane). Any residual erythrocytes were removed via ammonium-chloride-potassium (ACK) Lysing Buffer (Lonza) treatment for 60 sec at room temperature (RT). To assess PBMCs vitality, cells were stained with viability dye (Zombie NIR; Biolegend) for 15 min at RT and Fc-block was performed with 1% human serum for 10 min at RT. PBMCs were then stained for 15 min at RT with fluorophore-conjugated antibodies. Finally, cells were washed in 2% fetal bovine serum/PBS and live CD45pos/HLA-DRpos/lineageneg (CD3, CD19, CD56) were immediately FACS sorted on a FACSAria III (BD Biosciences). Cells resuspended in 0.5 ml PBS 1X plus 0.04% BSA were washed once by centrifugation and counted with an automatic cell counter (ThermoFisher; Countess II). About 20.000 cells per sample were loaded into one channel of the Chromium Chip B using the Chromium Single Cell 3' Reagent Kits (v3 Chemistry) (10X Genomics). 50 ng per sample of the barcoded and amplified cDNA was used then for constructing the sequencing libraries. Sequencing was performed on the NextSeq550 Illumina Platform, generating on average about 477 million reads per sample (on average about 67.000 reads per single cell recovered) following the configuration of the sequencing RUN indicated by the Single Cell 3' scRNAseq v3 protocol.