GSM7421515: Post-RTX remission, donor 2, scRNAseq; Homo sapiens; RNA-Seq
PBMC were isolated from fresh blood by Ficoll-paque density centrifugation and cryopreserved in FBS supplemented with 10% of DMSO. When all samples had been collected, PBMC were thawed at 37°C, treated with DNase I for 15 minutes, washed with RPMI containing 5% FBS and rested for 2 hours in a 37°C 5% CO2 incubator. Following rest, cells were washed twice with ice-cold PBS containing 2% FBS and stained with anti-human CD19 PerCp-Cy5.5 (BD Biosciences), anti-human CD4 Alexa Fluor 700 (BD Biosciences), and anti-human CD8α V500 (BD Biosciences) for 15 minutes on ice. Cell were washed again with ice-cold PBS containing 2% FBS, filtered through a 40 µm mesh, and a maximum of 50,000 live B cells were FACS isolated as CD19+ CD4- CD8- cells and on the basis of size and granularity. Sorted cells were then washed twice with ice-cold PBS containing 0.04% BSA (0.22 µm filtered) and brought to a concentration of 1,000 cells/µl. Samples were subsequently processed according to the 10x Genomics Single Cell 5' v1.1 user guide to target 10,000 events. Briefly, single cell PBMC suspensions were loaded onto the 10x Single Cell Chip G along with 10x Genomics NextGem scRNA 5' V1.1 reagents. Complementary DNA (cDNA) and 5' gene expression libraries were generated using the standard 10x Genomics protocol. 5' gene expression libraries were constructed with 10X Genomics Chromium Single Cell 5' v1.1 Reagent Kits following the manufacturer's standard protocols.