GSM7873660: Parse, H2, replicate1, scRNA-seq; Homo sapiens; RNA-Seq
Frozen vials of PBMCs were rapidly thawed in a 37°C water bath until pea-sized frozen cryopreserved cells remained. Thawed PBMCs were quenched by adding 12 mL 37°C pre-warmed 10% human serum media (Human Serum, Sigma Aldrich, # H4522; RPMI 1640 Medium, Gibco, # 11875-093; Penicillin-Streptomycin-Glutamine, Gibco, # 10378-016) and were centrifuged at 500 x g for 5 min at room temperature. The cell pellet was resuspended in 0.5 mL of pre-warmed human serum media and rested in the 37°C, 5% CO2 incubator for 30 mins. After resting, cell solutions were passed through a 40 𝞵m Falcon cell strainer (Thermo Fisher, # 08-771-1), and then centrifuged. Single live PBMCs were selected using fluorescence-activated cell sorting (FACS) with gating based on forward scatter (FSC), side scatter (SSC), and DAPI dye (Thermo Fisher, #D1306). The purified PBMCs were then resuspended with 1 mL 1X PBS containing 1% bovine serum albumin (BSA, Sigma Aldrich, # B2518). For the 10x Genomics platform, the cell count for each sample was determined using the TC20™ Automated Cell Counter (Sigma Aldrich). Approximately 16,000 cells per sample were used for library construction, utilizing the Chromium Next GEM Automated Single Cell 3ʹ Reagent Kits (10x Genomics). For the Parse Biosciences platform, the cells were prepared using the Fixation Kit (Parse Biosciences) according to the manufacturer's protocol. After fixation, the cell count was determined using the TC20™ Automated Cell Counter (Sigma Aldrich), resulting in an average of approximately 35,500 cells per donor. These cells were subjected to the Single Cell Whole Transcriptome Kit v2 (Parse Biosciences) for library construction.