A novel strand-specific RNA-sequencing protocol using dU-adaptor-assembled Tn5
A novel strand-specific RNA-sequencing protocol using dU-adaptor-assembled Tn5
复制标题
使用 dU 适配器组装的 Tn5 的新型链特异性 RNA 测序方案
DOI：
10.1093/jxb/erac515
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发表时间：
2022
影响因子：
6.9
通讯作者：
Shengchun Xu
中科院分区：
文献类型：
--
作者：
Xiaoyuan Tao;Shouli Feng;Sujuan Li;Guang Chen;Jian Wang;Lizhi Xu;Xujun Fu;Jing Yu;Shengchun Xu
摘要链特异性RNA-seq是发现新转录本、基因组注释和基因表达水平分析的有力工具。Tn 5转座酶已成功应用于大规模测序项目;特别是，Tn 5衔接子修饰用于表观遗传学，基因组结构和染色质可视化。我们开发了一种新的dU接头组装Tn 5介导的链特异性RNA测序方案，并将该方法与领先的dUTP方法在实验程序和所生成文库的多个质量指标方面进行了比较。结果表明，dU-Tn 5方法易于操作，并且考虑到文库复杂性、链特异性、均匀性和注释的转录物覆盖的连续性，产生具有可比质量的链特异性RNA-seq文库。我们还评估了dU-Tn 5方法在鉴定大豆根中氮响应蛋白编码基因和长非编码RNA中的性能。结果表明，约62-70%的差异表达基因（DEG）从传统的文库中检测到的dU-Tn 5文库中也被检测到，表明我们的方法与当前标准的良好协议;此外，他们的倍数变化是高度相关的（R 0.9）。因此，我们的方法提供了一个有前途的“自己动手”的基因表达谱的链RNA-seq程序。
SUMMARY. Strand-specific RNA-seq is a powerful tool for discovery of novel transcripts, annotation of genomes, and profiling of gene expression levels. Tn5 transposase has been successfully applied in massive-scale sequencing projects; in particular, Tn5 adaptor modification is used in epigenetics, genomic structure, and chromatin visualization. We developed a novel dU-adaptor-assembled Tn5-mediated strand-specific RNA-sequencing protocol and compared this method with the leading dUTP method in terms of experimental procedure and multiple quality metrics of the generated libraries. The results showed the dU-Tn5 method is easy to operate and generates a strand-specific RNA-seq library of comparable quality considering library complexity, strand-specificity, evenness, and continuity of annotated transcript coverage. We also evaluated the performance of the dU-Tn5 method in identifying nitrogen-responsive protein-coding genes and long non-coding RNAs in soybean roots. The results indicated that ~62-70% of differentially expressed genes (DEGs) detected from conventional libraries were also detected in dU-Tn5 libraries, indicating good agreement of our method with the current standard; moreover, their fold-changes were highly correlated (R 0.9). Thus, our method provides a promising “do-it-yourself” stranded RNA-seq procedure for gene expression profiling.