Nucleofector 96 well electroporation system

For CSR, a derivative of mouse B cell lymphoma line (CH12F3-2A) expressing Bcl2 was used throughout the study (29 (link)) unless stated. Cells were cultured and maintained in RPMI 1640 supplemented with glutamine, NCTC, fetal bovine serum (10%), β-mercaptoethanol, and penicillin/streptomycin. For KD experiments, the Silencer/Stealth siRNA or control (low GC) oligonucleotides were purchase from Thermo Fisher Scientific, MA, USA, and the ASO (control or target) were purchased from QIAGEN and were introduced into the cells using the Nucleofector 96-well electroporation system (Lonza, Switzerland) as described by the manufacturer’s instructions. After the transfection, the cells were cultured for 24 hours, then were stimulated by CIT cocktail (29 (link)) (anti-CD40L, IL-4, and TGF-β) or OHT (1 mM) for AIDER cells to induce IgM to IgA isotype switching, and cultured for another 24 to 48 hours before collection. The surface expression of IgM and IgA was examined by staining the cells with fluorescein isothiocyanate–conjugated anti-mouse IgM (eBioscience) and PE-conjugated anti-mouse IgA (eBioscience). Propidium iodide staining was included to stain the dead cells. The fluorescence-activated cell sorting (FACS) analysis was performed with a BD FACSCalibur instrument, and the data were analyzed by CellQuest software (BD Biosciences). The sequences of siRNA oligos are shown in table S2.