26 March 2024

Phospho-RNA sequencing with circAID-p-seq

Del Piano A, Kecman T, Schmid M, Barbieri R, Brocchieri L, Tornaletti S, Firrito C, Minati L, Bernabo P, Signoria I, Lauria F, Gillingwater TH, Viero G, Clamer M. Phospho-RNA sequencing with circAID-p-seq. Nucleic Acids Res. 2022 Feb 28;50(4):e23. doi: 10.1093/nar/gkab1158. PMID: 34850942; PMCID: PMC8887461.

Most RNA footprinting approaches that require ribonuclease cleavage generate RNA fragments bearing a phosphate or cyclic phosphate group at their 3' end. Unfortunately, current library preparation protocols rely only on a 3' hydroxyl group for adaptor ligation or poly-A tailing. Here, we developed circAID-p-seq, a PCR-free library preparation for selective 3' phospho-RNA sequencing. As a proof of concept, we applied circAID-p-seq to ribosome profiling, which is based on sequencing of RNA fragments protected by ribosomes after endonuclease digestion. CircAID-p-seq, combined with the dedicated computational pipeline circAidMe, facilitates accurate, fast and highly efficient sequencing of phospho-RNA fragments from eukaryotic cells and tissues. We used circAID-p-seq to portray ribosome occupancy in transcripts, providing a versatile and PCR-free strategy to possibly unravel any endogenous 3'-phospho RNA molecules.

Keywords: 3' phospho-RNA, Ribosome profiling, Phospho-RNA fragments, library preparation