Selective Amplification and Sequencing of Cyclic Phosphate-Containing RNAs by the cP-RNA-seq Method

Nat Protoc. 2016 Mar;11(3):476-89. doi: 10.1038/nprot.2016.025. Epub 2016 Feb 11.

Abstract

RNA digestions catalyzed by many ribonucleases generate RNA fragments that contain a 2',3'-cyclic phosphate (cP) at their 3' termini. However, standard RNA-seq methods are unable to accurately capture cP-containing RNAs because the cP inhibits the adapter ligation reaction. We recently developed a method named cP-RNA-seq that is able to selectively amplify and sequence cP-containing RNAs. Here we describe the cP-RNA-seq protocol in which the 3' termini of all RNAs, except those containing a cP, are cleaved through a periodate treatment after phosphatase treatment; hence, subsequent adapter ligation and cDNA amplification steps are exclusively applied to cP-containing RNAs. cP-RNA-seq takes ∼6 d, excluding the time required for sequencing and bioinformatics analyses, which are not covered in detail in this protocol. Biochemical validation of the existence of cP in the identified RNAs takes ∼3 d. Even though the cP-RNA-seq method was developed to identify angiogenin-generating 5'-tRNA halves as a proof of principle, the method should be applicable to global identification of cP-containing RNA repertoires in various transcriptomes.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line, Tumor
  • DNA, Complementary / genetics
  • High-Throughput Nucleotide Sequencing / methods*
  • Humans
  • Phosphates / analysis*
  • Phosphates / metabolism
  • RNA / chemistry
  • RNA / genetics*
  • RNA, Transfer / chemistry
  • RNA, Transfer / genetics
  • Ribonuclease, Pancreatic / genetics
  • Sequence Analysis, RNA / methods*

Substances

  • DNA, Complementary
  • Phosphates
  • RNA
  • RNA, Transfer
  • angiogenin
  • Ribonuclease, Pancreatic