Ingolia Nicholas T
Department of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute, University of California, San Francisco, CA, USA.
Methods Enzymol. 2010;470:119-42. doi: 10.1016/S0076-6879(10)70006-9. Epub 2010 Mar 1.
We present a detailed protocol for ribosome profiling, an approach that we developed to make comprehensive and quantitative measurements of translation in yeast. In this technique, ribosome positions are determined from their nuclease footprint on their mRNA template and the footprints are quantified by deep sequencing. Ribosome profiling has already enabled highly reproducible measurements of translational control. Because this technique reports on the exact position of ribosomes, it also revealed the presence of ribosomes on upstream open reading frames and demonstrated that ribosome density was higher near the beginning of protein-coding genes. Here, we describe nuclease digestion conditions that produce uniform ~28 nucleotide (nt) protected fragments of mRNA templates that indicate the exact position of translating ribosomes. We also give a protocol for converting these RNA fragments into a DNA library that can be sequenced using the Illumina Genome Analyzer. Unbiased conversion of anonymous, small RNAs into a sequencing library is challenging, and we discuss standards that played a key role in optimizing library generation. Finally, we discuss how deep sequencing data can be used to quantify gene expression at the level of translation.
我们展示了一种核糖体谱分析的详细方案，这是我们开发的一种用于对酵母中的翻译进行全面定量测量的方法。在这项技术中，核糖体的位置是根据其在mRNA模板上的核酸酶足迹来确定的，并且这些足迹通过深度测序进行定量。核糖体谱分析已经能够实现对翻译调控的高度可重复测量。由于这项技术报告了核糖体的确切位置，它还揭示了上游开放阅读框上核糖体的存在，并证明了在蛋白质编码基因起始处附近核糖体密度更高。在这里，我们描述了核酸酶消化条件，该条件可产生均匀的约28个核苷酸（nt）的受保护mRNA模板片段，这些片段指示了正在翻译的核糖体的确切位置。我们还给出了一个将这些RNA片段转化为可使用Illumina基因组分析仪进行测序的DNA文库的方案。将匿名的小RNA无偏差地转化为测序文库具有挑战性，我们讨论了在优化文库生成中起关键作用的标准。最后，我们讨论了如何使用深度测序数据在翻译水平上定量基因表达。
