Phospho-RNA sequencing with CircAID-p-seq

by Piano, Alessia Del; Barbieri, Ruggero; Schmid, Michael; Brocchieri, Luciano; Tornaletti, Silvia; Firrito, Claudia; Minati, Luca; Bernabo, Paola; Signoria, Ilaria; Lauria, Fabio; Gillingwater, Thomas H.; Viero, Gabriella and Clamer, Massimiliano
Abstract:
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.
Reference:
Phospho-RNA sequencing with CircAID-p-seq (Piano, Alessia Del; Barbieri, Ruggero; Schmid, Michael; Brocchieri, Luciano; Tornaletti, Silvia; Firrito, Claudia; Minati, Luca; Bernabo, Paola; Signoria, Ilaria; Lauria, Fabio; Gillingwater, Thomas H.; Viero, Gabriella and Clamer, Massimiliano), In bioRxiv, Cold Spring Harbor Laboratory, 2020.
Bibtex Entry:
@article {Piano2020.12.16.423093,
	author = {Piano, Alessia Del and Barbieri, Ruggero and Schmid, Michael and Brocchieri, Luciano and Tornaletti, Silvia and Firrito, Claudia and Minati, Luca and Bernabo, Paola and Signoria, Ilaria and Lauria, Fabio and Gillingwater, Thomas H. and Viero, Gabriella and Clamer, Massimiliano},
	title = {Phospho-RNA sequencing with CircAID-p-seq},
	elocation-id = {2020.12.16.423093},
	year = {2020},
	doi = {10.1101/2020.12.16.423093},
	publisher = {Cold Spring Harbor Laboratory},
	abstract = {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.},
	URL = {https://www.biorxiv.org/content/early/2020/12/16/2020.12.16.423093},
	eprint = {https://www.biorxiv.org/content/early/2020/12/16/2020.12.16.423093.full.pdf},
	journal = {bioRxiv}
}