Phospho-RNA sequencing with circAID-p-seq

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.

Automated summary

We developed a PCR-free library preparation method called circAID-p-seq for selective 3' phospho-RNA sequencing. By applying it to ribosome profiling, we were able to accurately and quickly sequence phospho-RNA fragments from eukaryotic cells and tissues, and depict ribosome occupancy in transcripts.