Puromycin

Viral supernatants were produced after transfecting 293 T cells with pGag-pol, pVSV-G, and individual expression vectors (pSUPER-puro, pSUPER-puro-shSIRT1, and pSUPER-puro-shSIRT3) using the HilyMax reagent (Dojindo) as previously described [25 (link)]. The cells were cultured at 37°C in DMEM supplemented with 10% FBS for 24 h. Medium was replaced with fresh DMEM supplemented with 2% FBS and incubated for an additional 24 h. Viral supernatant was collected and supplemented with 10 mg/mL polybrene (Merck Millipore). The target cells were infected with this viral supernatant for 24 h at 37°C. After infection, the cells were selected with 3 μg/mL puromycin (Enzo Life Sciences, Farmingdale, NY) for 3 days.

To generate a stable cell line, the cells were transfected with a NOX1 shRNA plasmid vector (sc-156079-SH, Santa Cruz, Santa Cruz, CA, USA). After 72 h of transfection, the cells were selected using diluted 10 μg/mL of puromycin (Enzo Biochem, Farmingdale, NY, USA) in the culture media.

BJAB and BJAB-TACI cells deficient for BAFFR were generated by lentiviral transduction of a CRISPR/Cas9-expression vector carrying a hBAFFR gRNA (Supplementary Table 5). Annealed oligonucleotides 5ʹ-CACCGGGCCGAGTGCTTCGACCTGC-3ʹ and 5ʹ-AAACGCAGGTCGAAGCACTCGGCCC-3ʹ were cloned in the BsmBI restriction site of lentrcrispr v2 plasmid (Supplementary Table 5)^{48 (link)}. This plasmid was co-transfected with co-vectors pCMV-VSV-g and psPAX2 (Supplementary Table 5) into 293 T cells with polyethyleneimide. The next day, cells were washed with PBS and cultured for an additional day in RPMI 10% FCS. Cell supernatants filtered at 0.45 µm were supplemented with 8 µg/ml polybrene (Sigma, H9268) and 3 ml were added to 2 × 10⁶ pelleted target cells that were subsequently cultured overnight in a 12-well plate, then expanded for 2 days in a 6-well plate. Cells were then submitted to two rounds of selection in RPMI 10% FCS, 1 µg/ml puromycin (EnzoLifeSciences). Surviving cells were cloned in RPMI 10% FCS. BAFFR-deficient clones were identified by flow cytometry after staining with biotinylated huBR9.1 at 2 µg/ml, followed by PE-coupled streptavidin at 1/500.

H1563 cells were transfected (Neon electroporator (Thermo Fisher Scientific) with TRIP13 CRISPR Activation Plasmid (sc-404006-ACT; Santa Cruz Biotechnology) or Control CRISPR plasmid (sc-437275; Santa Cruz Biotechnology). After 48 h, transfected cells were selected using 1ug/mL puromycin (Enzo Life Sciences, ALX-380-028). TRIP13 overexpression was screened by Q-PCR and Western blot analysis.