Heyer Erin E, Ozadam Hakan, Ricci Emiliano P, Cenik Can, Moore Melissa J
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA.
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA.
Nucleic Acids Res. 2015 Jan;43(1):e2. doi: 10.1093/nar/gku1235. Epub 2014 Dec 12.
Deep sequencing of strand-specific cDNA libraries is now a ubiquitous tool for identifying and quantifying RNAs in diverse sample types. The accuracy of conclusions drawn from these analyses depends on precise and quantitative conversion of the RNA sample into a DNA library suitable for sequencing. Here, we describe an optimized method of preparing strand-specific RNA deep sequencing libraries from small RNAs and variably sized RNA fragments obtained from ribonucleoprotein particle footprinting experiments or fragmentation of long RNAs. Our approach works across a wide range of input amounts (400 pg to 200 ng), is easy to follow and produces a library in 2-3 days at relatively low reagent cost, all while giving the user complete control over every step. Because all enzymatic reactions were optimized and driven to apparent completion, sequence diversity and species abundance in the input sample are well preserved.
链特异性cDNA文库的深度测序如今已成为一种广泛应用的工具，用于鉴定和定量各种样本类型中的RNA。从这些分析得出的结论的准确性取决于将RNA样本精确且定量地转化为适合测序的DNA文库。在此，我们描述了一种优化方法，可从小RNA以及从核糖核蛋白颗粒足迹实验或长RNA片段化获得的大小可变的RNA片段制备链特异性RNA深度测序文库。我们的方法适用于广泛的输入量（400 pg至200 ng），易于操作，能以相对较低的试剂成本在2 - 3天内生成文库，同时让用户对每一步都有完全的掌控。由于所有酶促反应都经过优化并趋向于明显完成，输入样本中的序列多样性和物种丰度得以很好地保留。
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