Chotewutmontri Prakitchai, Stiffler Nicholas, Watkins Kenneth P, Barkan Alice
Department of Biology, Institute of Molecular Biology, University of Oregon, Eugene, OR, 97403, USA.
Methods Mol Biol. 2018;1676:165-183. doi: 10.1007/978-1-4939-7315-6_10.
Ribosome profiling (also known as Ribo-seq) provides a genome-wide, high-resolution, and quantitative accounting of mRNA segments that are occupied by ribosomes in vivo. The method has been used to address numerous questions in bacteria, yeast, and metazoa, but its application to questions in plant biology is just beginning. This chapter provides a detailed protocol for profiling ribosomes in plant leaf tissue. The method was developed and optimized with maize, but it has been used successfully with Arabidopsis and tobacco as well. The method captures ribosome footprints from the chloroplast and cytosol in the same preparation, but it is not optimal for detecting the footprints of mitochondrial ribosomes. The protocol is robust and simpler than many of the methods reported previously for ribosome profiling in plants.
核糖体谱分析（也称为核糖体测序）可在全基因组范围内对体内被核糖体占据的mRNA片段进行高分辨率定量分析。该方法已被用于解决细菌、酵母和后生动物中的诸多问题，但其在植物生物学问题中的应用才刚刚起步。本章提供了一份用于分析植物叶片组织中核糖体的详细方案。该方法是针对玉米开发并优化的，但也已成功应用于拟南芥和烟草。该方法在同一制备过程中可捕获叶绿体和细胞质中的核糖体足迹，但对于检测线粒体核糖体的足迹并非最佳选择。该方案稳健且比之前报道的许多植物核糖体谱分析方法更简单。
