A streamlined ribosome profiling protocol for the characterization of microorganisms
A streamlined ribosome profiling protocol for the characterization of microorganisms
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DOI：
10.2144/000114302
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发表时间：
2015-06-01
期刊：
影响因子：
2.7
通讯作者：
Palsson, Bernhard O.
中科院分区：
文献类型：
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作者：
Latif, Haythem;Szubin, Richard;Palsson, Bernhard O.
核糖体分析是在基因组规模上表征体内蛋白质翻译的有力工具，具有从详细的分子机制到系统级预测建模的多种应用。虽然非常有效，但这种复杂的技术尚未在微生物研究界得到广泛应用。在这里，我们提出了一个简化的核糖体分析协议，降低了进入微生物表征研究的门槛。我们的方法在收获、裂解和回收单体期间提供了简化的替代方案，并且还消除了几个耗时的步骤，特别是在文库构建期间的大小选择步骤。此外，最终文库中rRNA和tRNA的丰度急剧降低。我们简化的工作流程可实现更高的吞吐量，将从收获到最终文库的时间缩短一半（缩短至3-4天），并生成高比例的信息读取，同时保持现有方案的高质量标准。
Ribosome profiling is a powerful tool for characterizing in vivo protein translation at the genome scale, with multiple applications ranging from detailed molecular mechanisms to systems-level predictive modeling. Though highly effective, this intricate technique has yet to become widely used in the microbial research community. Here we present a streamlined ribosome profiling protocol with reduced barriers to entry for microbial characterization studies. Our approach provides simplified alternatives during harvest, lysis, and recovery of monosomes and also eliminates several time-consuming steps, in particular size-selection steps during library construction. Furthermore, the abundance of rRNAs and tRNAs in the final library is drastically reduced. Our streamlined workflow enables greater throughput, cuts the time from harvest to the final library in half (down to 3-4 days), and generates a high fraction of informative reads, all while retaining the high quality standards of the existing protocol.