Authors: Alessia Del Piano, Ruggero Barbieri, Michael Schmid, Luciano Brocchieri, Silvia Tornaletti, Claudia Firrito, Luca Minati, Paola Bernabo, Ilaria Signoria, Fabio Lauria, Thomas H. Gillingwater, Gabriella Viero, Massimiliano Clamer.
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.
Read more on: https://nanoporetech.com/resource-centre/phospho-rna-sequencing-circaid-p-seq