Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba, Japan.
Division of Biological Science, Graduate School of Science and Technology, Nara Institute of Science and Technology, Nara, Japan.
Nucleic Acids Res. 2024 Jan 25;52(2):e7. doi: 10.1093/nar/gkad1116.
Precise detection of the transcriptional start site (TSS) is a key for characterizing transcriptional regulation of genes and for annotation of newly sequenced genomes. Here, we describe the development of an improved method, designated 'TSS-seq2.' This method is an iterative improvement of TSS-seq, a previously published enzymatic cap-structure conversion method to detect TSSs in base sequences. By modifying the original procedure, including by introducing split ligation at the key cap-selection step, the yield and the accuracy of the reaction has been substantially improved. For example, TSS-seq2 can be conducted using as little as 5 ng of total RNA with an overall accuracy of 96%; this yield a less-biased and more precise detection of TSS. We then applied TSS-seq2 for TSS analysis of four plant species that had not yet been analyzed by any previous TSS method.
精确检测转录起始位点（TSS）是研究基因转录调控和新测序基因组注释的关键。在这里，我们描述了一种改进方法的开发，称为“TSS-seq2”。该方法是对先前发表的酶促帽结构转化方法 TSS-seq 的迭代改进，用于在碱基序列中检测 TSS。通过修改原始程序，包括在关键的帽选择步骤中引入拆分连接，反应的产量和准确性得到了极大的提高。例如，TSS-seq2 可以使用低至 5ng 的总 RNA 进行，总体准确性为 96%；这可以更准确地检测 TSS，且结果偏差更小。然后，我们将 TSS-seq2 应用于尚未通过任何先前 TSS 方法进行分析的四个植物物种的 TSS 分析。
