Shore Sabrina, Henderson Jordana M, McCaffrey Anton P
Research and Development, Cell and Molecular Biology, TriLink BioTechnologies, LLC, San Diego, CA, USA.
Methods Mol Biol. 2018;1712:145-161. doi: 10.1007/978-1-4939-7514-3_10.
Next-generation small RNA sequencing is a valuable tool which is increasing our knowledge regarding small noncoding RNAs and their function in regulating genetic information. Library preparation protocols for small RNA have thus far been restricted due to higher RNA input requirements (>10 ng), long workflows, and tedious manual gel purifications. Small RNA library preparation methods focus largely on the prevention or depletion of a side product known as adapter dimer that tends to dominate the reaction. Adapter dimer is the ligation of two adapters to one another without an intervening library RNA insert or any useful sequencing information. The amplification of this side reaction is favored over the amplification of tagged library since it is shorter. The small size discrepancy between these two species makes separation and purification of the tagged library very difficult. Adapter dimer hinders the use of low input samples and the ability to automate the workflow so we introduce an improved library preparation protocol which uses chemically modified adapters (CleanTag) to significantly reduce the adapter dimer. CleanTag small RNA library preparation workflow decreases adapter dimer to allow for ultra-low input samples (down to approx. 10 pg total RNA), elimination of the gel purification step, and automation. We demonstrate how to carry out this streamlined protocol to improve NGS data quality and allow for the use of sample types with limited RNA material.
下一代小RNA测序是一种有价值的工具，它正在增加我们对小非编码RNA及其在调控遗传信息中功能的了解。由于对RNA输入量要求较高（>10 ng）、工作流程长以及繁琐的手动凝胶纯化，小RNA文库制备方案至今仍受到限制。小RNA文库制备方法主要集中在预防或去除一种称为接头二聚体的副产物，这种副产物往往在反应中占主导地位。接头二聚体是两个接头相互连接，中间没有文库RNA插入片段或任何有用的测序信息。由于这种副反应产物较短，其扩增比带标签文库的扩增更占优势。这两种产物之间的大小差异使得带标签文库的分离和纯化非常困难。接头二聚体阻碍了低输入量样本的使用以及工作流程的自动化，因此我们引入了一种改进的文库制备方案，该方案使用化学修饰的接头（CleanTag）来显著减少接头二聚体。CleanTag小RNA文库制备工作流程减少了接头二聚体，从而允许使用超低输入量样本（总RNA低至约10 pg），省去了凝胶纯化步骤，并实现了自动化。我们展示了如何执行这个简化方案来提高NGS数据质量，并允许使用RNA材料有限的样本类型。
