MeRIP-Seq Aag2 cells Infected with DENV
Total RNA was fragmented using the RNA Fragmentation Reagent (Thermo Fisher) for 15 min. MeRIP was performed using EpiMark N6-methyladenosine Enrichment kit (NEB). 25 mL Protein G Dynabeads (Thermo Fisher) per sample were washed three times in MeRIP buffer (150 mM NaCl, 10 mM Tris-HCl [pH 7.5], 0.1% NP-40), and incubated with 1 mL anti-m6A antibody for 2 h at 4 with rotation. After washing three times with MeRIP buffer, anti-m6A conjugated beads were incubated with purified mRNAs with rotation at 4 overnight in 300 mL MeRIP buffer with 1 mL RNase inhibitor (recombinant RNasin; Promega). 10% of the mRNA sample was saved as the input fraction. Beads were then washed twice with 500 mL MeRIP buffer, twice with low salt wash buffer (50 mM NaCl, 10 mM Tris-HCl [pH 7.5], 0.1% NP-40), twice with high salt wash buffer (500 mM NaCl, 10 mM Tris-HCl [pH 7.5],0.1% NP-40), and once again with MeRIP buffer. m6A-modified RNAs were eluted twice in 100 mL of MeRIP buffer containing 5 mM m6A salt (Santa Cruz Biotechnology) for 30 min at 4 with rotation. Eluates were pooled and concentrated by ethanol precipitation.