PT  - JOURNAL ARTICLE
AU  - Alessia Del Piano
AU  - Ruggero Barbieri
AU  - Michael Schmid
AU  - Luciano Brocchieri
AU  - Silvia Tornaletti
AU  - Claudia Firrito
AU  - Luca Minati
AU  - Paola Bernabo
AU  - Ilaria Signoria
AU  - Fabio Lauria
AU  - Thomas H. Gillingwater
AU  - Gabriella Viero
AU  - Massimiliano Clamer
TI  - Phospho-RNA sequencing with CircAID-p-seq
AID  - 10.1101/2020.12.16.423093
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2020.12.16.423093
4099  - http://biorxiv.org/content/early/2020/12/16/2020.12.16.423093.short
4100  - http://biorxiv.org/content/early/2020/12/16/2020.12.16.423093.full
AB  - Accurate positional information concerning ribosomes and RNA binding proteins with respect to their transcripts is important to understand the global regulatory network underlying protein and RNA fate in living cells. Most footprinting approaches generate RNA fragments bearing a phosphate or cyclic phosphate groups at their 3′ end. Unfortunately, all current protocols for library preparation rely only on the presence of a 3′ hydroxyl group. Here, we developed circAID-p-seq, a PCR-free library preparation for 3′ phospho-RNA sequencing. We applied circAID-p-seq to ribosome profiling, which produces fragments protected by ribosomes after endonuclease digestion. CircAID-p-seq, combined with the dedicated computational pipeline circAidMe, facilitates accurate, fast, highly efficient and low-cost sequencing of phospho-RNA fragments from eukaryotic cells and tissues. While assessing circAID-p-seq to portray ribosomes engaged with transcripts, we provide a versatile tool to unravel any 3′-phospho RNA molecules.Competing Interest StatementM.C. is the founder of, director of, and a shareholder in IMMAGINA BioTechnology S.r.l., a company engaged in the development of new technologies for gene expression analysis at the ribosomal level. L.M, A.D.P., C.F., P.B are employees of IMMAGINA BioTechnology S.r.l. G.V is a scientific advisor of IMMAGINA BioTechnology S.r.l. All of the other authors declare no competing interests. CircAID-p-seq technology is covered by the international patent application number PCT/IB2020/058259.