Illumina NovaSeq 6000 paired end sequencing; Single Cell Analysis of B cells in COVID-19 comprising active and recovered disease
Single Cell Analysis of B cells in COVID-19 comprising active and recovered disease
Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood by density gradient centrifugation with Biocoll cell separation solution (Biochrom AG, Berlin, Germany) and frozen in FCS + 10% DMSO. PBMCs were thawed and washed twice with PBS. One million cells were stained with smCD3-APC-H7 (clone SK7, BD), CD19-PC7 (clone J3-119, Beckman Coulter) and CD20-V450 (clone 2H7, BioLegend) for 30 min at room temperature. Flow cytometry was performed on a BD FACSLyricTM. Untouched B cells were isolated from PBMCs via magnetic separation using the B Cell Isolation Kit II (Miltenyi). Viability ranged between 92-98% as determined with the Cell Viability Analyzer Vi-Cell XR (Beckman Coulter).   After in vitro staining with APC anti-CD19 (BD) and amcyan live/dead stain, viable CD19+ cells were sorted with a 70 μm nozzle into PBS plus 0.1% BSA at a concentration of 100 cells/μl. Cells were processed within 1 h after collection according to 5´ GEX and V(D)J protocols suggested by 10x. 5´ GEX and V(D)J protocols suggested by 10x