Protocol for differential analysis of pseudouridine modifications using nanopore DRS and unmodified transcriptome control

In this protocol, we detect pseudouridine using nanopore direct RNA sequencing (DRS 002/004) and analyze dynamic changes to mRNA modifications in response to perturbation. We cover RNA extraction, poly(A) selection, DRS, unmodified transcriptome (in vitro transcribed [IVT]) library preparation, and knockdowns of writer proteins as critical controls. We detail preprocessing, psi-site identification, and differential analysis, quantifying the direction and magnitude of changes across cellular types and states. For complete details on the use and execution of this protocol, please refer to Fanari et al.¹.