GSM8305841: PBMCs from patient 3 and 4 with Mock and EV treatment, scRNAseq_pool2; Homo sapiens; RNA-Seq
PBMCs analyzed in flow cytometry and gene expression experiments were isolated from mixed anticoagulated blood of 3 healthy volunteers by adding 10 volumes of 1× red blood cell (RBC) lysis buffer (8.02 g l-1 NH4Cl, 0.84 g l-1 NaHCO3, 0.37 g l-1 EDTA), incubate for 10 – 15 min at room temperature on a shaker at 90 rpm. Stop the reaction by adding 20 – 30 volumes of 1× PBS. PBMCs were pelleted at 400 g for 5 min at 4 °C, washed with 1× PBS, and resuspended in RPMI-1640 with 10% heat-inactivated FBS and 100 U ml-1 penicillin and streptomycin for culture or in 90% FBS and 10% DMSO for long-term storage in liquid nitrogen. The fixed cells were next subjected to 4-multiplexed probe hybridization, single-cell gel beads in the emulsion (GEM) generation and barcoding, GEM recovery and pre-amplification, fixed RNA-Gene expression library construction and finally sequencing using Chromium fixed RNA profiling reagent kits for multiplexed samples (10x Genomics, PN-1000415, PN-1000420, PN-1000421) and Chromium next GEM chip Q single cell kit (10x Genomics, PN-1000422) according to the manufacturer's protocol.