Purification of rgRSV particles that are naturally defective in m6A

RSV particles that are naturally defective in m⁶A were purified using our established protocol [79]. Ten T150 flasks of wild type U2OS cells and METTL3-knockdown U2OS cells were infected with rgRSV at an MOI of 0.01. When extensive cytopathic effects (CPE) were observed, cell culture supernatants were harvested and clarified by centrifugation at 10,000 × g for 30 min. Virus was concentrated through a 35% (wt/vol) sucrose cushion by centrifugation at 30,000 × g for 2 h at 4°C in a Ty 50.2 rotor (Beckman, Brea, CA). The pellet was resuspended in NTE buffer (0.05 M Tris-HCl, 0.15 M NaCl, 15 mM CaCl2, pH 6.5). Virus was further purified through 30–50% sucrose gradient ultracentrifugation. The band containing virus particles were collected and centrifuged at 25,000 × g for 2 h at 4°C. The pellet was resuspended in DMEM with 10% trehalose.