Elmer Katharina, Helfer Stephanie, Mirkovic-Hösle Milijana, Förstemann Klaus
Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, D-81377, Munich, Germany.
Methods Mol Biol. 2014;1173:33-49. doi: 10.1007/978-1-4939-0931-5_4.
The small RNA silencing system is an important player in the control of gene expression. In particular analysis of the highly complex sequence repertoire of piRNAs and endogenous siRNAs directed against repetitive elements and transposons has been facilitated by the advent of next-generation sequencing tools. We are providing a detailed protocol for the creation of deep sequencing libraries in combination with a chemical modification step (periodate oxidation and β-elimination) that enriches for 3'-terminal modified small RNAs. This step can thus facilitate the detection and-since incorporation of the small RNA into the effector complex precedes the terminal methylation-determine the Argonaute-loading state of certain small RNA species. Both, the oxidation as well as the deep sequencing library preparation are adaptations of popular, previously published protocols. The sequencing libraries described here can be sequenced on all flow cell types (i.e., single-end and paired-end) of the Illumina sequencing systems and are compatible with Illumina index reads. It is therefore also possible to analyze the libraries on the MiSeq system, for which currently only paired-end flow cells are available, and combine several libraries in multiplexed experiments. The chapter concludes with considerations concerning quality control and data analysis.
小RNA沉默系统是基因表达调控中的重要参与者。特别是，新一代测序工具的出现促进了对针对重复元件和转座子的piRNA和内源性siRNA高度复杂序列库的分析。我们提供了一个详细的方案，用于创建深度测序文库，并结合化学修饰步骤（高碘酸盐氧化和β-消除），该步骤可富集3'-末端修饰的小RNA。因此，这一步骤有助于检测，并且由于小RNA掺入效应复合物先于末端甲基化，所以可以确定某些小RNA种类的AGO装载状态。氧化以及深度测序文库制备都是对先前发表的流行方案的改编。这里描述的测序文库可以在Illumina测序系统的所有流动池类型（即单端和双端）上进行测序，并且与Illumina索引读取兼容。因此，也可以在MiSeq系统上分析文库，目前该系统仅提供双端流动池，并在多重实验中合并多个文库。本章最后讨论了质量控制和数据分析相关问题。
