Department of Biochemistry and Cell Biology, Max F. Perutz Laboratories, University of Vienna, Austria.
Methods. 2010 Oct;52(2):125-32. doi: 10.1016/j.ymeth.2010.06.004. Epub 2010 Jun 9.
Genomic SELEX is a discovery tool for genomic aptamers, which are genomically encoded functional domains in nucleic acid molecules that recognize and bind specific ligands. When combined with genomic libraries and using RNA-binding proteins as baits, Genomic SELEX used with high-throughput sequencing enables the discovery of genomic RNA aptamers and the identification of RNA-protein interaction networks. Here we describe how to construct and analyze genomic libraries, how to choose baits for selections, how to perform the selection procedure and finally how to analyze the enriched sequences derived from deep sequencing. As a control procedure, we recommend performing a "Neutral" SELEX experiment in parallel to the selection, omitting the selection step. This control experiment provides a background signal for comparison with the positively selected pool. We also recommend deep sequencing the initial library in order to facilitate the final in silico analysis of enrichment with respect to the initial levels. Counter selection procedures, using modified or inactive baits, allow strengthening the binding specificity of the winning selected sequences.
基因组 SELEX 是一种用于基因组适体的发现工具，基因组适体是核酸分子中基因组编码的功能域，能够识别和结合特定的配体。当与基因组文库结合并使用 RNA 结合蛋白作为诱饵时，高通量测序与基因组 SELEX 相结合可以发现基因组 RNA 适体并鉴定 RNA-蛋白质相互作用网络。在这里，我们描述了如何构建和分析基因组文库，如何选择用于选择的诱饵，如何进行选择程序，以及如何最终分析来自深度测序的富集序列。作为对照程序，我们建议在选择中并行进行“中性”SELEX 实验，省略选择步骤。此对照实验提供了与阳性选择池进行比较的背景信号。我们还建议对初始文库进行深度测序，以便于最终针对初始水平进行富集的计算机分析。使用修饰或失活的诱饵进行反选择程序可以增强获胜选择序列的结合特异性。
