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improved 24 . Nanopore sequencing methods were designed to directly detect RNA modifications based on base-calling errors, which are bioinformatically demanding and highly sensitive to the abundance of the transcripts as well as modification levels. Hence, a quantitative, accurate and sensitive method for global Ψ detection is urgently needed.

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Pseudouridine (ψ) is an abundant post-transcriptional RNA modification in ncRNA and mRNA. However, transcriptome-wide measurement of individual ψ sites remains unaddressed. Here, we develop "PRAISE", via selective chemical labeling of ψ by bisulfite to induce nucleotide deletion signature during reverse transcription, to realize quantitative assessment of the ψ landscape in the human transcriptome. Unlike traditional RNA/DNA bisulfite treatment, our approach is based on quaternary base mapping and revealed a ~10% median modification level for 2,714 confident ψ sites in HEK293T cells. By perturbing pseudouridine synthases, we obtained differential mRNA targets of PUS1, PUS7 and TRUB1, with TRUB1 mRNA targets showing the highest modification stoichiometry. In addition, we identified and quantified known and novel ψ sites in mitochondrial mRNA, catalyzed by a mitochondria-localized isoform of PUS1. Collectively, we provide a sensitive and convenient method to measure transcriptome-wide ψ ; we envision this quantitative approach would facilitate emerging efforts to elucidate the function and mechanism of mRNA pseudouridylation.