There are three major techniques for the site-specific mapping of pseudouridine in RNA, called Pseudo-seq, Ψ-seq and PSI-seq. All these methods are based on the unique reaction between pseudouridine and N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMCT). The RNA to be analyzed is fragmented and incubated with CMCT. Even if CMCT can form covalent bonds with U, G and Ψ residues, only Ψ-CMC is resistant to alkaline hydrolysis (U-CMC and G-CMC get hydrolyzed). Next, reverse transcription is done to obtain a cDNA library, with the cDNAs terminating one nucleotide downstream the pseudouridine residue. Next generation sequencing of the cDNA library will indicate where the modified pseudouridine residue is located in the RNA. In order to do this, two cDNA libraries are prepared, one in which the RNA has undergone CMC treatment and the other one without CMC treatment. Differences in the length of the reads between the two libraries will indicate where the Ψ residues are. Another method is called CeU-Seq, which uses a biotinylated derivative of CMCT. This enables the purification and enrichment of biotinylated transcripts (transcripts modified with pseudouridine) with streptavidin columns, therefore reducing the library size and increasing sensitivity.
Other pseudouridine detection methods include site-specific cleavage and radioactive-labeling followed by ligation-assisted extraction and thin-layer chromatography (SCARLET) and mass spectrometry.