Lyu Jun, Chen Chongyi
Laboratory of Biochemistry and Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.
Bio Protoc. 2024 Jun 5;14(11):e4998. doi: 10.21769/BioProtoc.4998.
Single-cell RNA sequencing (scRNA-seq) stands as a cutting-edge technology widely used in biological and biomedical research. Existing scRNA-seq methods rely on reverse transcription (RT) and second-strand synthesis (SSS) to convert RNA to cDNA before amplification. However, these methods often suffer from limited RT/SSS efficiency, which compromises the sensitivity of RNA detection. Here, we develop a new method, linearly amplified single-stranded RNA-derived transcriptome sequencing (LAST-seq), which directly amplifies the original single-stranded RNA without prior RT and SSS and offers high-sensitivity RNA detection and a low level of technical noise in single-cell transcriptome analysis. LAST-seq has been applied to quantify transcriptional bursting kinetics in human cells, advancing our understanding of chromatin organization's role in regulating gene expression. Key features • An RNase H/DNA polymerase-based strategy to attach the T7 promoter to single-stranded RNA. • T7 promoter mediated IVT on single stranded RNA template at single cell level.
单细胞RNA测序（scRNA-seq）是一项前沿技术，广泛应用于生物学和生物医学研究。现有的scRNA-seq方法在扩增前依靠逆转录（RT）和第二链合成（SSS）将RNA转化为cDNA。然而，这些方法的RT/SSS效率往往有限，这会影响RNA检测的灵敏度。在此，我们开发了一种新方法，即线性扩增单链RNA衍生转录组测序（LAST-seq），该方法无需事先进行RT和SSS即可直接扩增原始单链RNA，并在单细胞转录组分析中提供高灵敏度的RNA检测和低水平的技术噪音。LAST-seq已应用于定量人类细胞中的转录爆发动力学，加深了我们对染色质组织在调节基因表达中作用的理解。关键特性•基于核糖核酸酶H/DNA聚合酶的策略，将T7启动子连接到单链RNA上。•在单细胞水平上，T7启动子介导单链RNA模板上的体外转录。
