Cloning and Identification of Recombinant Argonaute Bound Small RNAs Using Next-Generation Sequencing
Cloning and Identification of Recombinant Argonaute Bound Small RNAs Using Next-Generation Sequencing
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DOI：
10.1007/978-1-4939-7339-2_1
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发表时间：
2018-01-01
期刊：
影响因子：
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通讯作者：
Singh, Guramrit
中科院分区：
文献类型：
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作者：
Gangras, Pooja;Dayeh, Daniel M.;Singh, Guramrit
Argonaute蛋白（AGO）装载有小RNA作为识别靶mRNA的向导。由于靶特异性在很大程度上取决于两条链之间的碱基互补性，因此鉴定与AGO结合的小向导RNA和长靶RNA是重要的。为此，下一代测序（NGS）技术将我们的理解真正扩展到了核苷酸水平。然而，通过NGS从稀缺的RNA样品中鉴定RNA仍然是一个挑战。此外，大多数商业和公开的方法与小RNA或长RNA兼容，但不等同地适用于两者。因此，产生定量的、无偏倚的NGS文库以从低水平输入中鉴定小的和长的RNA的单一方法将受到广泛关注。在这里，我们介绍这样一个程序，是基于两个已发表的协议的几个修改，并允许强大的，灵敏的，可重复的克隆和测序的少量的RNA的可变长度。该方法被应用于鉴定结合到纯化的真核AGO的小RNA。在将DNA衔接子连接到RNA 3 '端之后，该方法的关键特征是使用衔接子来引发逆转录（RT），其中生物素化的脱氧核糖核苷酸特异性地掺入延伸的互补DNA中。这样的RT产物在链霉亲和素珠上富集，在固定在珠上的同时环化，并直接用于PCR扩增。我们提供了一个逐步指南，以生成RNA-Seq文库，它们的纯化，定量，验证和下一代测序的准备。我们还提供了使用Galaxy进行NGS后数据分析的基本步骤，Galaxy是一个开源的基于网络的平台。
Argonaute proteins (AGOs) are loaded with small RNAs as guides to recognize target mRNAs. Since the target specificity heavily depends on the base complementarity between two strands, it is important to identify small guide and long target RNAs bound to AGOs. For this purpose, next-generation sequencing (NGS) technologies have extended our appreciation truly to the nucleotide level. However, the identification of RNAs via NGS from scarce RNA samples remains a challenge. Further, most commercial and published methods are compatible with either small RNAs or long RNAs, but are not equally applicable to both. Therefore, a single method that yields quantitative, bias-free NGS libraries to identify small and long RNAs from low levels of input will be of wide interest. Here, we introduce such a procedure that is based on several modifications of two published protocols and allows robust, sensitive, and reproducible cloning and sequencing of small amounts of RNAs of variable lengths. The method was applied to the identification of small RNAs bound to a purified eukaryotic AGO. Following ligation of a DNA adapter to RNA 3'-end, the key feature of this method is to use the adapter for priming reverse transcription (RT) wherein biotinylated deoxyribonucleotides specifically incorporated into the extended complementary DNA. Such RT products are enriched on streptavidin beads, circularized while immobilized on beads and directly used for PCR amplification. We provide a stepwise guide to generate RNA-Seq libraries, their purification, quantification, validation, and preparation for next-generation sequencing. We also provide basic steps in post-NGS data analyses using Galaxy, an open-source, web-based platform.