GSM6430184: scRNA-seq - Patient 8; Homo sapiens; RNA-Seq
Frozen PBMCs were thawed in a 37°C water bath for 3-5 min until no ice was visible. Cells were washed twice with 1 mL PBS + 0.04% BSA and pelleted (300×g for 5 min at 4°C). Dead cells were removed according to the Demonstrated Protocol: Removal of Dead Cells from Single Cell Suspensions for Single Cell RNA Sequencing (10X Genomics). Using the MACS Dead Cell Removal Kit (Miltenyi Biotec), the pellet was resuspended in 100 mL Dead Cell Removal MicroBeads and incubated for 15 minutes at room temperature. After incubation, the cell suspension was diluted with 1X Binding Buffer and applied to a MS column. The dead cells were retained on the column and the live cells passed through the column and were collected. After dead cell removal, the samples were washed twice with 1 mL PBS + 0.04% BSA and the cell concentration was determined using a Cellometer K2 cell counter (Nexcelom Biosciences). The cells were then aliquoted for scRNA-seq. The cells were first counted and measured for viability using either the Vi-Cell XR Cell Viability Analyzer (Beckman-Coulter, Brea, CA) or a basic hemocytometer and light microscope. The barcoded Gel Beads were thawed from -80°C and the cDNA master mix was prepared according to the manufacturer's instruction for Chromium Next GEM Single Cell 3' Library and Gel Bead Kit (10x Genomics, Pleasanton, CA). Based on the desired number of cells to be captured for each sample, a volume of live cells was mixed with the cDNA master mix. A per sample concentration of 400,000 cells per milliliter or better is required for the standard targeted cell recovery of approximately 4000 cells. The stock concentration requirements would not change for higher cell recovery numbers. The cell suspension and master mix, thawed Gel Beads and partitioning oil were added to a Chromium Single Cell G chip. The filled chip was loaded into the Chromium Controller, where each sample was processed and the individual cells within the sample were captured into uniquely labeled GEMs (Gel Beads-In-Emulsion). The GEMs were collected from the chip and taken to the bench for reverse transcription, GEM dissolution, and cDNA clean-up. The resulting cDNA contained a pool of uniquely barcoded molecules. A portion of the cleaned and measured pooled cDNA continued to library construction, where standard Illumina sequencing primers and a 10x Genomics unique i7 Sample index were added to each cDNA pool. All cDNA pools and resulting libraries were measured using Qubit High Sensitivity assays (Thermo Fisher Scientific, Waltham, MA) and Agilent Bioanalyzer High Sensitivity chips (Agilent, Santa Clara, CA).