Putative pseudouridine residues in alphavirus RNA derived from human and mosquito cells
We infected human U2OS cells and mosquito C7/10 cells with SINV or CHIKV (MOI=5), alongside mock infections, to identify putative pseudouridine residues in viral and host RNA. U2OS cells were harvested at 8 hours post-infection (SINV) or 18 hours post-infection (CHIKV), while C7/10 cells were harvested at 48 hours post-infection for both viruses. We used Psi-seq, a sequencing technique in which CMC selectively modifies pseudouridine in RNA, followed by base treatment. Each sample was either CMC-treated or mock-treated and included an internal control RNA containing a single pseudouridine to validate CMC treatment efficacy. This modification causes reverse transcriptase to terminate one base downstream of pseudouridine sites during library preparation, enabling identification of putative pseudouridine residues by comparing CMC+ to CMC- libraries.