Single B Cell Transcriptome Profiling in NMOSD

Paired VH:VL sequencing of single B cells from patients with NMOSD was carried out as previously described.^{26 (link)} Briefly, total B cells were isolated from PBMCs using the Human Memory B Cell Isolation Kit (Miltenyi Biotec) with an LD column. The B cells were loaded into a syringe at 2 × 10⁵ cells/mL. All the reagents required for overlap extension (OE) RT-PCR was loaded into a second syringe. The syringes were connected to a Y junction and simultaneously expel into the emulsification oil. The emulsion was aliquoted into 96-well plates at 100 μL/well, and OE RT-PCR was performed. VH:VL amplicons were then extracted from the emulsions, amplified using nested PCR, and sequenced by 2 × 300 Illumina MiSeq, as previously described.^{25 (link)}