Hoerth Katharina, Reitter Sonja, Schott Johanna
Division of Biochemistry, Mannheim Institute for Innate Immunoscience, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany.
Center for Molecular Biology of Heidelberg University (ZMBH), German Cancer Research Center (DKFZ)-ZMBH Alliance, Heidelberg, Germany.
Bio Protoc. 2022 Feb 20;12(4):e4323. doi: 10.21769/BioProtoc.4323.
Ribosome profiling (Ribo-Seq) is a highly sensitive method to quantify ribosome occupancies along individual mRNAs on a genome-wide scale. Hereby, ribosome-protected fragments (= footprints) are generated by nuclease digestion, isolated, and sequenced together with the corresponding randomly fragmented input samples, to determine ribosome densities (RD). For library preparation, equal amounts of total RNA are used. Subsequently, all transcript fragments are subjected to linker ligation, cDNA synthesis, and PCR amplification. Importantly, the number of reads obtained for every transcript in input and footprint samples during sequencing depends on sequencing depth and library size, as well as the relative abundance of the transcript in the sample. However, the information pertaining to the absolute amount of input and footprint sequences is lost during sample preparation, hence ruling out any conclusion whether translation is generally suppressed or activated in one condition over the other. Therefore, the RD fold-changes determined for individual genes do not reflect absolute regulation, but have to be interpreted as relative to bulk mRNA translation. Here, we modified the original ribosome profiling protocol that was first established by Ingolia (2009), by adding small amounts of yeast lysate to the mammalian lysates of interest as a spike-in. This allows us to not only detect changes in the RD of specific transcripts relative to each other, but also to simultaneously measure global differences in RD (normalized ribosome density values) between samples. Graphic abstract: Ribo-Seq measures changes in RD of specific mRNAs relative to bulk mRNA. The addition of a yeast-lysate, as a spike-in for normalization of read counts, allows for an absolute measurement of changes in RD.
核糖体谱分析（Ribo-Seq）是一种高度灵敏的方法，可在全基因组范围内定量单个mRNA上的核糖体占有率。在此过程中，通过核酸酶消化产生核糖体保护片段（即足迹），将其分离，并与相应的随机片段化输入样本一起测序，以确定核糖体密度（RD）。文库制备时，使用等量的总RNA。随后，所有转录本片段都要进行接头连接、cDNA合成和PCR扩增。重要的是，测序过程中输入样本和足迹样本中每个转录本获得的读数数量取决于测序深度和文库大小，以及样本中转录本的相对丰度。然而，在样本制备过程中，与输入序列和足迹序列绝对量相关的信息丢失了，因此无法得出在一种条件下翻译是普遍受到抑制还是激活的结论。因此，为单个基因确定的RD倍数变化并不反映绝对调控，而必须解释为相对于整体mRNA翻译的相对变化。在这里，我们修改了最初由英戈利亚（2009年）建立的核糖体谱分析方案，在感兴趣的哺乳动物裂解物中加入少量酵母裂解物作为内参。这使我们不仅能够检测特定转录本的RD相对于彼此的变化，还能同时测量样本之间RD的整体差异（标准化核糖体密度值）。图形摘要：Ribo-Seq测量特定mRNA的RD相对于整体mRNA的变化。添加酵母裂解物作为读数计数标准化的内参，可对RD变化进行绝对测量。
