Nuclease-mediated depletion biases in ribosome footprint profiling libraries

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 abundant, ribosomal RNA sequences in order to save on sequencing costs. The most effective commercial solution (Ribo-Zero) has been discontinued 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 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. Overall design: Ribosome profiling libraries were genereated in several cell lines using different rRNA depletion techniques

核糖体印迹测序（Ribosome footprint profiling）是一种基于高通量测序的技术，可实现活细胞内翻译过程的精细化、全景式解析。该技术的核心环节之一是去除冗余且丰度极高的核糖体RNA序列，以降低测序成本。此前最有效的商业化解决方案（Ribo-Zero）已停产，随后市场上涌现出多款实验原理各异的新型商业化应用方案。本研究对多款针对人类样本RNA测序（RNA-seq）开发的商业化替代方案进行了评估，结果显示其均不适用于核糖体印迹测序。因此我们推荐使用早期核糖体印迹测序研究中广泛应用的定制化生物素标记寡核苷酸探针。尤为重要的是，我们提醒：基于靶向核酸酶切割的核糖体RNA去除方案会显著干扰可从数据中获取的高分辨率信息，因此对于需要精准确定RNA片段末端的所有应用场景，均不建议使用此类方案。
整体实验设计：研究采用多种核糖体RNA去除技术，在多个细胞系中构建了核糖体印迹测序文库。