Selective amplification and sequencing of cyclic phosphate-containing RNAs by the cP-RNA-seq method.
Selective amplification and sequencing of cyclic phosphate-containing RNAs by the cP-RNA-seq method.
复制标题
DOI：
10.1038/nprot.2016.025
复制
发表时间：
2016-03
期刊：
影响因子：
14.8
通讯作者：
Kirino Y
中科院分区：
文献类型：
--
作者：
Honda S;Morichika K;Kirino Y
许多核糖核酸酶催化的RNA双链反应产生3′-末端含有2′，3 ′-环磷酸（cP）的RNA片段。然而，标准RNA-seq方法不能准确捕获含cP的RNA，因为cP抑制衔接子连接反应。我们最近开发了一种名为“cP-RNA-seq”的方法，该方法能够选择性地扩增和测序含cP的RNA。在这里，我们描述了cP-RNA-seq方案，其中除了含有cP的RNA之外，所有RNA的3′-末端在磷酸酶处理后通过高碘酸盐处理被切割，因此随后的接头连接和cDNA扩增步骤仅适用于含有cP的RNA。cP-RNA-seq需要约6天，不包括测序和生物信息学分析所需的时间，本方案中未详细介绍此类下游测定。在鉴定的RNA中存在cP的生物化学验证需要约3天。尽管cP-RNA-seq方法是为了鉴定产生血管生成素的5′-tRNA半体而开发的，但该方法应适用于各种转录组中含cP的RNA库的全局鉴定。
RNA digestions catalyzed by many ribonucleases generate RNA fragments containing 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, such downstream assays 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.