通过 mim-tRNAseq 分析真核细胞 tRNA 池的实验和计算工作流程。
Experimental and computational workflow for the analysis of tRNA pools from eukaryotic cells by mim-tRNAseq.
机构信息
Mechanisms of Protein Biogenesis, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany.
Mechanisms of Protein Biogenesis, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany; Department of Chemistry, Technical University of Munich, 85748 Garching, Germany.
出版信息
STAR Protoc. 2022 Jul 31;3(3):101579. doi: 10.1016/j.xpro.2022.101579. eCollection 2022 Sep 16.
Quantifying tRNAs is crucial for understanding how they regulate mRNA translation but is hampered by their extensive sequence similarity and premature termination of reverse transcription at multiple modified nucleotides. Here, we describe the use of modification-induced misincorporation tRNA sequencing (mim-tRNAseq), which overcomes these limitations with optimized library construction and a comprehensive toolkit for data analysis and visualization. We outline algorithm improvements that enhance the efficiency and accuracy of read alignment and provide details on data analysis outputs using example datasets. For complete details on the use and execution of this protocol, please refer to Behrens et al. (2021).
定量 tRNA 对于理解它们如何调节 mRNA 翻译至关重要，但由于其广泛的序列相似性和在多个修饰核苷酸处的逆转录过早终止，这一任务受到了阻碍。在这里，我们描述了使用修饰诱导的错配 tRNA 测序（mim-tRNAseq），它通过优化的文库构建和全面的数据分析和可视化工具包克服了这些限制。我们概述了算法改进，这些改进提高了读取比对的效率和准确性，并提供了使用示例数据集的数据分析输出的详细信息。有关该协议的使用和执行的完整详细信息，请参阅 Behrens 等人（2021 年）。
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