Matts Jessica A, Sytnikova Yuliya, Chirn Gung-Wei, Igloi Gabor L, Lau Nelson C
Department of Biology, Rosenstiel Basic Medical Science Research Center, Brandeis University, Waltham, MA, USA.
Methods Mol Biol. 2014;1093:123-36. doi: 10.1007/978-1-62703-694-8_10.
Increasingly, the discovery and characterization of small regulatory RNAs from a variety of organisms have all required deep-sequencing methodologies. However, the crux to successful deep-sequencing analysis depends upon optimal construction of a cDNA library compatible for the high-throughput sequencing platform. Challenges to small RNA library constructions arise when dealing with minute tissue samples because certain structural RNA fragments can dominate and mask the desired characterization of regulatory small RNAs like microRNAs (miRNAs), endogenous small interfering RNAs (endo-siRNAs), and Piwi-interacting RNAs (piRNAs). Here, we describe methods that improve the chances of constructing a successful library from small RNAs isolated from minute tissues such as enriched follicle cells from the Drosophila ovarium. Because the ribosomal RNA (rRNA) fragments are frequently the major contaminants in small RNA preparations from minute amounts of tissue, we demonstrate the utility of antisense oligonucleotide depletion and an acryloylaminophenylboronic acid (APB) polyacrylamide gel system for separating the abundant 2S rRNA in Drosophila from endo-siRNAs and piRNAs. Finally, our methodology generates libraries amenable to multiplex sequencing on the Illumina Hi-Seq platform.
越来越多的情况是，从各种生物体中发现和鉴定小调节RNA都需要深度测序方法。然而，深度测序分析成功的关键取决于与高通量测序平台兼容的cDNA文库的最佳构建。当处理微量组织样本时，小RNA文库构建会面临挑战，因为某些结构性RNA片段可能占主导并掩盖了诸如微小RNA（miRNA）、内源性小干扰RNA（endo-siRNA）和Piwi相互作用RNA（piRNA）等调节性小RNA的所需特征。在这里，我们描述了一些方法，这些方法提高了从微小组织（例如果蝇卵巢中富集的卵泡细胞）分离的小RNA构建成功文库的几率。由于核糖体RNA（rRNA）片段通常是微量组织小RNA制备中的主要污染物，我们展示了反义寡核苷酸去除法和丙烯酰氨基苯硼酸（APB）聚丙烯酰胺凝胶系统在从果蝇的内源性小干扰RNA和Piwi相互作用RNA中分离丰富的2S rRNA方面的效用。最后，我们的方法生成了适合在Illumina Hi-Seq平台上进行多重测序的文库。
