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Selective amplification and sequencing of cyclic phosphate–containing RNAs by the cP-RNA-seq method

Nature Protocols volume 11, pages 476–489 (2016)Cite this article

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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.

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Figure 1: A schematic representation of the reactivity of a 3′-P and a 3′-cP with enzymatic treatments.
Figure 2: A schematic representation of the cP-RNA-seq procedure for selective amplification and sequencing of cP-containing RNAs.
Figure 3: Selective amplification of mouse 5′-tRNA halves using cP-RNA-seq.
Figure 4: Biochemical validation of the presence of a cP in a mouse 5′-tRNA half.

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Acknowledgements

This study was supported by a US National Institutes of Health (NIH) grant (GM106047, to Y.K.) and a Japan Society for the Promotion of Science (JSPS) Postdoctoral Fellowships for Research Abroad (to S.H.).

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  1. Computational Medicine Center, Sidney Kimmel Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania, USA

    Shozo Honda, Keisuke Morichika & Yohei Kirino

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  1. Shozo Honda
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Contributions

S.H. and Y.K. conceived cP-RNA-seq and the general experimental design. S.H. and K.M. performed experiments. S.H., K.M. and Y.K. wrote the paper.

Corresponding author

Correspondence to Yohei Kirino.

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The authors declare no competing financial interests.

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Honda, S., Morichika, K. & Kirino, Y. Selective amplification and sequencing of cyclic phosphate–containing RNAs by the cP-RNA-seq method. Nat Protoc 11, 476–489 (2016). https://doi.org/10.1038/nprot.2016.025

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