EBNA3C Knockout and Rev Virus Production for Primary B Cell Isolation

Recombinant EBNA3C KO and Rev viruses, virus production, and primary B cell isolation have been described previously (Anderton et al., 2008 (link); Skalska et al., 2013 (link)). B cell purity was assessed to be >90% CD20⁺ using anti–CD20-APC (eBioscience) staining and flow cytometric analysis. RNA was harvested from uninfected primary B cells. Primary B cells (from anonymous donor buffy coats, purchased from the UK Blood Transfusion Service) were infected with virus-containing supernatant, and infected cells were incubated in RPMI 1640 (Thermo Fisher Scientific) supplemented with 15% fetal bovine serum, penicillin, and streptomycin at 37°C and 5% CO₂. Over a period of 30 d, infected cells were harvested for RNA extraction and replaced by fresh medium every 3 or 5 d.