Phospho-RNA sequencing with circAID-p-seq

Nucleic Acids Res. 2022 Feb 28;50(4):e23. doi: 10.1093/nar/gkab1158.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Gene Library
  • High-Throughput Nucleotide Sequencing / methods
  • Phosphates
  • RNA* / genetics
  • Ribosomes* / genetics
  • Sequence Analysis, RNA / methods

Substances

  • Phosphates
  • RNA