Howard Hughes Medical Institute (HHMI).
Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
RNA. 2020 Oct;26(10):1481-1488. doi: 10.1261/rna.075523.120. Epub 2020 Jun 5.
Ribosome footprint profiling is a high-throughput sequencing-based technique that provides detailed and global views of translation in living cells. An essential part of this technology is removal of unwanted, normally very abundant, ribosomal RNA sequences that dominate libraries and increase sequencing costs. The most effective commercial solution (Ribo-Zero) has been discontinued as a standalone product and a number of new, experimentally distinct commercial applications have emerged on the market. Here we evaluated several commercially available alternatives designed for RNA-seq of human samples and find them generally unsuitable for ribosome footprint profiling. We instead recommend the use of custom-designed biotinylated oligos, which were widely used in early ribosome profiling studies. Importantly, we warn that depletion solutions based on targeted nuclease cleavage significantly perturb the high-resolution information that can be derived from the data, and thus do not recommend their use for any applications that require precise determination of the ends of RNA fragments.
核糖体足迹分析是一种基于高通量测序的技术，可提供活细胞中翻译的详细和全局视图。该技术的一个重要部分是去除通常非常丰富的不需要的核糖体 RNA 序列，这些序列会主导文库并增加测序成本。最有效的商业解决方案（Ribo-Zero）已不再作为独立产品销售，市场上出现了许多新的、实验性不同的商业应用。在这里，我们评估了几种专为人类样本 RNA-seq 设计的商业上可获得的替代品，发现它们通常不适合核糖体足迹分析。相反，我们建议使用广泛用于早期核糖体分析研究的定制设计的生物素化寡核苷酸。重要的是，我们警告说，基于靶向核酸酶切割的消耗解决方案会严重干扰可以从数据中得出的高分辨率信息，因此不建议将其用于任何需要精确确定 RNA 片段末端的应用。
