Lu Bo, Yi Chengqi
State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, China.
Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China.
Curr Protoc. 2023 Apr;3(4):e735. doi: 10.1002/cpz1.735.
Tn5 transposase has been widely used to simultaneously fragment and tag double-stranded DNA (dsDNA) with sequencing adaptors in library construction for next-generation sequencing. Recently, we demonstrated that Tn5 transposase also possesses tagmentation activity toward RNA/DNA hybrids, in addition to its canonical dsDNA substrates. Based on this new activity, we are able to skip multiple laborious and time-consuming steps in traditional RNA-seq methods, and rapid, low-input, cost-effective, one-tube RNA-seq library construction is thus enabled. The libraries constructed by Transposase-assisted RNA/DNA hybrids Co-tagmEntation (termed "TRACE-seq") demonstrate excellent performance in terms of gene expression measurement and differential gene expression analysis. Here, we present detailed protocols for TRACE-seq that will be broadly useful for the study of RNA biology and biomedical research. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Total RNA preparation Basic Protocol 2: TRACE-seq library construction Support Protocol: Tn5 transposome assembly.
Tn5转座酶已被广泛用于在二代测序文库构建中，用测序接头同时对双链DNA（dsDNA）进行片段化和标记。最近，我们证明Tn5转座酶除了对其经典的dsDNA底物具有活性外，对RNA/DNA杂交体也具有转座标签活性。基于这一新活性，我们能够跳过传统RNA测序方法中多个费力且耗时的步骤，从而实现快速、低起始量、经济高效的单管RNA测序文库构建。通过转座酶辅助的RNA/DNA杂交体共转座标签法（称为“TRACE-seq”）构建的文库在基因表达测量和差异基因表达分析方面表现出色。在此，我们展示了TRACE-seq的详细方案，这将对RNA生物学研究和生物医学研究广泛有用。© 2023威利期刊有限责任公司。基本方案1：总RNA制备 基本方案2：TRACE-seq文库构建 支持方案：Tn5转座体组装
