GSM7152961: B3, scBCR; Homo sapiens; RNA-Seq
Peripheral blood mononuclear cells (PBMC) of different donors were isolated by the standard density-gradient centrifugation and cryopreserved until the day of use.  The CD19+ cell fraction was enriched from PBMCs by positive selection with CD19 magnetic microbeads (Miltenyi Biotech) and subsequently stained on ice for 20 min with the following fluorochrome-labeled mouse monoclonal antibodies: CD20 (clone 2H7, BioLegend) and APC mouse anti-human IgG (Clone: G18-145 BD Pharmingen). Cells were sorted to over 99% purity on a FACSAria III (BD). FACS-sorted cells were collected in 6 μl FCS in Eppendorf tubes that were pre-coated overnight with 2% BSA. Memory B cells were targeted for single-cell RNA-seq analysis using the BD Rhapsody Single-Cell Analysis System (BD Biosciences; Fan et al., 2015). Briefly, the single-cell suspension was loaded into a BD Rhapsody cartridge with >200,000 microwells, and single-cell capture was achieved by random distribution and gravity precipitation. Next, the bead library was loaded into the microwell cartridge to saturation so that the bead was paired with a cell in a microwell. The cells were lysed in a microwell cartridge to hybridize mRNA molecules onto bar-coded capture oligos on the beads. These beads were then retrieved from the microwell cartridge into a single tube for subsequent cDNA synthesis, exonuclease I digestion, and multiplex-PCR–based library construction. Sequencing was performed on NovaSeq paired-end mode.