A robust method for measuring aminoacylation through tRNA-Seq.
A robust method for measuring aminoacylation through tRNA-Seq.
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一种通过 tRNA-Seq 测量氨酰化的可靠方法。
DOI：
10.1101/2023.07.31.551363
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发表时间：
2024
期刊：
影响因子：
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通讯作者：
Sullivan,LucasB
中科院分区：
文献类型：
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作者：
Davidsen,Kristian;Sullivan,LucasB
目前定量氨基酰化tRNAs比例的方法，也称为tRNA电荷，受到产量、精密度和/或准确度较低的问题的限制。在这里，我们提出了一种优化的电荷转移RNA测序(tRNA-Seq)方法，该方法结合了以前的发展和新描述的方法，以建立一种精确和准确的tRNA电荷测量的协议。我们验证了该协议提供了稳健的tRNA氨基酰化的量化，并且我们提供了一种端到端的方法，可以扩展到数百个样本，包括用于数据处理的软件。此外，我们还表明，该方法支持相对tRNA表达水平的测量，并可用于通过反转录误结合来推断tRNA修饰，从而支持tRNA生物学中的多用途应用。
Current methods to quantify the fraction of aminoacylated tRNAs, also known as the tRNA charge, are limited by issues with either low throughput, precision, and/or accuracy. Here, we present an optimized charge transfer RNA sequencing (tRNA-Seq) method that combines previous developments with newly described approaches to establish a protocol for precise and accurate tRNA charge measurements. We verify that this protocol provides robust quantification of tRNA aminoacylation and we provide an end-to-end method that scales to hundreds of samples including software for data processing. Additionally, we show that this method supports measurements of relative tRNA expression levels and can be used to infer tRNA modifications through reverse transcription misincorporations, thereby supporting multipurpose applications in tRNA biology.