ABSTRACT

In conventional RNA high-throughput sequencing, modified bases prevent a large fraction of tRNA transcripts to be converted into cDNA libraries. Recent proposals aiming at resolving this issue take advantage of the interference of base modifications with RT enzymes to detect and identify them by establishing signals from aborted cDNA transcripts. Because some modifications, such as methyl groups, do almost not allow RT bypassing, demethylation and highly processive RT enzymes have been used to overcome these obstacles. Working with Escherichia coli as a model system, we show that with a conventional (albeit still engineered) RT enzyme and key optimizations in library preparation, all RT-impairing modifications can be highlighted along the entire tRNA length without demethylation procedure. This is achieved by combining deep-sequencing samples, which allows to establish aborted transcription signal of higher accuracy and reproducibility, with the potential for differentiating tiny differences in the state of modification of all cellular tRNAs. In addition, our protocol provides estimates of the relative tRNA abundance.

摘要

在传统的 RNA 高通量测序中，修饰的碱基会阻止大部分 tRNA 转录物转化为 cDNA 文库。最近旨在解决这个问题的提议利用碱基修饰对 RT 酶的干扰，通过从中止的 cDNA 转录物中建立信号来检测和识别它们。由于某些修饰（例如甲基）几乎不允许 RT 绕过，因此已使用去甲基化和高度持续性的 RT 酶来克服这些障碍。与大肠杆菌一起工作作为一个模型系统，我们展示了使用传统的（尽管仍然是工程化的）RT 酶和文库制备中的关键优化，可以沿着整个 tRNA 长度突出显示所有损害 RT 的修饰，而无需去甲基化程序。这是通过结合深度测序样本来实现的，该样本允许建立具有更高准确性和可重复性的中止转录信号，并具有区分所有细胞 tRNA 修饰状态的微小差异的潜力。此外，我们的协议提供了相对 tRNA 丰度的估计。