使用PacBio平台进行单分子多分子A尾部测序 (SM-PATseq)
James R Iben1, Tianwei Li2, Sandy Mattijssen3
1Molecular Genetics Core, Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institutes of Health (NIH), Bethesda, MD, USA. ibenjame@nih.gov.
Methods in molecular biology (Clifton, N.J.)
|October 12, 2023
概括
我们开发了SM-PATseq,这是一种用于核酸分辨率下测序整个转录组的多项 (A) 尾长度的新方法. 这种技术可以精确测量信使RNA多基解,这对于基因表达调节至关重要.
科学领域:
- 分子生物学分子生物学
- 基因组学就是基因组学.
- 在RNA生物学,RNA生物学.
背景情况:
- 传递 RNA (mRNA) 3'端的多基化是mRNA稳定性和翻译的关键调节者.
- 精确测量多种类型的尾巴长度对于理解基因表达调节至关重要.
研究的目的:
- 开发和描述一种新的单分子方法,用于在整个转录组中测序多分子 (A) 尾长.
- 通过使用长读序列测序技术,使多类 (A) 尾的核酸分辨率分析成为可能.
主要方法:
- 开发了单分子多分子A尾部测序 (SM-PATseq) 方法.
- 利用oligo-dT 3' splint适配器绑定进行第一链cDNA合成,然后随机六合体原始化进行第二链合成.
- 使用长读测序 (太平洋生物科学后续平台) 进行直接的cDNA测序.
主要成果:
- 在核酸分辨率下,SM-PATseq允许测定整个转录组的多元A尾长.
- 该方法可以在多A尾中识别非腺酸.
- 可以量化转录丰富度,类似于传统的RNA测序方法.
结论:
- SM-PATseq提供了一个强大的工具,用于高分辨率分析多种尾动力学.
- 这种方法有助于更深入地了解多基化在转录后基因调节中的作用.
- 描述的协议使得从总RNA中生成,测序和初级分析多个A尾数据.
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