GSM6815431: PBMC AGS6 patient n°1; Homo sapiens; RNA-Seq
Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation (2200 rpm or 974g without break for 30 minutes) using Ficoll (Eurobio Scientific, Les Ulis, France). After centrifugation, the pellet was resuspended in Phosphate-buffered saline (PBS) (Thermo Fisher scientific, Illkirch, France) and cells were centrifuged at 1500 rpm (453g)  for 5 minutes. Finally, the pellet was frozen in a medium containing 90% of Fetal Bovine Serum (FBS) (GIBCO, Thermo Fisher scientific, Illkirch, France) and 10% of dimethyl sulfoxide (DMSO) (Sigma Aldrich, St. Quentin Fallavier, France), and stored in liquid nitrogen.  PBMCs were thawed according to 10X Genomics protocol. The scRNA-seq libraries were generated using Chromium Single Cell 3′ Library & Gel Bead Kit v.1 (for AGSexp1 and AGSexp2) or v.3 (for AGSexp3) (10x Genomics) according to the manufacturer's protocol. Briefly, cells were counted, diluted at 1000 cells/µL in PBS+0,04% and 20,000 cells were loaded in the 10x Chromium Controller to generate single-cell gel-beads in emulsion. After reverse transcription, gel-beads in emulsion were disrupted. Barcoded complementary DNA was isolated and amplified by PCR. Following fragmentation, end repair and A-tailing, sample indexes were added during index PCR