GSM8218884: scRNA-seq_Dn1 PBMC of healthy donors; Homo sapiens; RNA-Seq
The peripheral blood mononuclear cells (PBMCs) were separated from whole blood by density gradient centrifugation using Lymphosep, Lymphocyte Separation Media (#L0560-500, Biowest, France), 1.077 g/ml density, at 600g for 30 minutes. PBMCs were washed twice with DPBS without calcium and magnesium at 300 g for 10 minutes and counted using CytoFLEX flow cytometer (Beckman Coulter, USA). 1 000 000 PBMCs were used to prepare cell suspension for single-cell sequencing. Cells were put into 1.5 ml cryo tubes and mixed with 500 μl of X-VIVO™ 10 Medium (#180989, Lonza, Switzerland), 400 μl of fetal bovine serum (#10500064, ThermoFisher Scientific, USA), and 100 μl of dimethyl sulfoxide (#F135, Paneco, Russia). Subsequently, the cell suspension was stored in cryo container at -80°C for 48 hours, and then stored in liquid nitrogen until further experiments (but not more than 6 months). Single-cell RNA sequencing was performed on the Chromium X platform, using the 10х Genomics Chromium Next GEM Single Cell 3′ Reagent Kit v3.1. (10х Genomics, USA). Prior to library preparation, cells were counted and quality of samples was assessed. Up to 8000 cells were used for further manipulations. cDNA amplification and library construction were conducted following the manufacturer's protocol.