GSM8341457: LuminalA_BC_115; Homo sapiens; RNA-Seq
Peripheral venous blood was collected into K2EDTA tubes (Vacutainer BD, Franklin Lakes, NJ, USA). The PBMCs were isolated using density gradient centrifugation with Lympholyte H Cell Separation Medium (Cedarlane, Burlington, ON, Canada) and SepMate 50 centrifuge tubes (StemCells Technologies, Vancouver, BC, Canada). The remaining platelets and erythrocytes were removed using the low-speed centrifugation and red blood cell lysis, respectively. The isolated PBMCs were stored in liquid nitrogen at a density of 1-10E6 cells/mL. A combination of 10% dimethyl sulfoxide (Sigma-Aldrich, Bur-lington, MA, USA) and 40% heat-inactivated fetal bovine serum (Gibco, ThermoFisher Scientific, Waltham, MA, USA) was used as the cryopreservation medium. The cell concentration was determined using the CytoSmart automated cell counter (Corning Inc., Corning, NY, USA). To perform scRNA-seq the 10X Chromium System (10X Genomics, Pleasanton, CA, USA) was used. First, the cryopreserved PBMCs were thawed and washed in Dulbecco's Phosphate-Buffered Saline supplemented with 5% FBS to remove the cryopreservation medium. The quality of the cell samples was confirmed using the trypan blue exclusion and viability of all samples was >90%. 2E6 cells per sample were used for the library preparation. RNA-seq was performed on 26 RNA samples extracted from PMBCs. Paired-end RNA-seq (oligo dT, stranded mRNA library, DNBseq platform, PE150, 20M reads/6Gb clean data) was performed at BGI (Hong Kong, China) using a NEBNext® Poly(A) mRNA Magnetic Isolation Module and BGI kit.