GSM8635071: N2-S-MB-BCR; Homo sapiens; OTHER
B cells were isolated from previously frozen PBMCs by immunomagnetic negative selection according to the manufacturer's protocol (EasySep Human B Cell Enrichment Kit, STEMCELL). The purified B cells were first stained with 1200 ng N1-M5 or N2-M5 probes (600 ng per color) for 30 min at 4 ˚C, then stained with CD20, CD19, CD27, IgD, and L/D blue. The live NA-specific MBCs were sorted into the FCS buffer and directly loaded on a 10X Chromium A chip. Single-cell lysis and cDNA synthesis were performed by using the 5' library and gel bead kit according to the provided protocol. The first-strand cDNA of 10X Barcoded was purified by silane magnetic beads, and sufficient mass was obtained by PCR amplification. The PCR products were quantified and qualified by Qubit3.0 Fluorometer (Invitrogen) and Agilent 2100 Bioanalyzer (Agilent). Then, the transcriptome and VDJ library preparation is performed by using 10X Chromium Single Cell VDJ Kits. All libraries were quantified by using Qsep100 (Bioptic) and Qubit3.0 Fluorometer (Invitrogen). Then libraries with different indices were multiplexed and loaded on an Illumina NovaSeq