title: Selective amplification and sequencing of cyclic phosphate-containing RNAs by the cP-RNA-seq method.
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.
authors: Computational Medicine Center, Sidney Kimmel Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania, USA.
authors: Shozo
authors: S
authors: Honda
authors: Computational Medicine Center, Sidney Kimmel Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania, USA.
authors: Keisuke
authors: K
authors: Morichika
authors: Computational Medicine Center, Sidney Kimmel Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania, USA.
authors: Yohei
authors: http://orcid.org/0000-0001-5232-4742
authors: Y
authors: Kirino
mesh_terms: D045744:Cell Line, Tumor; D018076:DNA, Complementary / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D010710:Phosphates / Q000032:analysis* / Q000378:metabolism; D012313:RNA / Q000737:chemistry / Q000235:genetics*; D012343:RNA, Transfer / Q000737:chemistry / Q000235:genetics; D012259:Ribonuclease, Pancreatic / Q000235:genetics; D017423:Sequence Analysis, RNA / Q000379:methods*
chemical_list: D018076:DNA, Complementary; D010710:Phosphates; D012313:RNA; D012343:RNA, Transfer; C046366:angiogenin; D012259:Ribonuclease, Pancreatic