Sequencing of First-strand cDNA Library Reveals Full-length Transcriptomes

We developed a new method of preparing libraries for strand-specific RNA sequencing (ssRNA-Seq). It employs Direct Ligation of Adaptors to First-strand cDNA (DLAF). We compared ssRNA-Seq libraries prepared using either the DLAF and dUTP methods from mouse embryonic stem cells (mES) and libraries were sequenced from one end or both ends. We also conducted a comparison of ssRNA-Seq libraries prepared using DLAF and ScriptSeq v2 kit (Epicenter) from mouse embryonic cortex (mECx). RNA was isolated using either Trizol or Qiagen Rneasy kit. rRNA is depleted using Eukaryote Ribominus v2 kit and libraries were prepared using one of the methods.

本研究开发了一种用于链特异性RNA测序（strand-specific RNA sequencing, ssRNA-Seq）的文库制备新方法，该方法依托接头与第一链cDNA直接连接法（Direct Ligation of Adaptors to First-strand cDNA, DLAF）。研究以小鼠胚胎干细胞（mouse embryonic stem cells, mES）为材料，对比了分别采用DLAF法与dUTP法制备的ssRNA-Seq文库，并对两类文库开展单端及双端测序。此外，本研究以小鼠胚胎皮层（mouse embryonic cortex, mECx）为材料，比较了采用DLAF法与Epicenter公司的ScriptSeq v2试剂盒制备的ssRNA-Seq文库。实验过程中，RNA提取分别使用TRIzol试剂或Qiagen RNeasy试剂盒，核糖体RNA去除采用真核生物核糖体RNA去除试剂盒v2（Eukaryote Ribominus v2 kit），最终通过上述任一方法完成文库构建。