ausblenden:
Schlagwörter:
Animals; Gene Expression Profiling/*methods; *Gene Library; High-Throughput Nucleotide Sequencing/*methods; Humans; Nucleic Acid Amplification Techniques; RNA, Messenger/isolation & purification
Zusammenfassung:
The analysis of transcriptome, which was over the past decade based mostly on microarray technologies, is now being superseded by so-called next generation sequencing (NGS) systems that changed the way to explore entire transcriptome. RNA sequencing (RNA-Seq), one application of NGS, is a powerful tool, providing information not only about the expression level of genes but also further about the structure of transcripts as it enables to unequivocally identify splicing events, RNA editing products, and mutations in expressed coding sequences within a single experiment. Herein, we describe step by step the deoxy-UTP (dUTP) strand-marking protocol [Parkhomchuk, D., Borodina, T., Amstislavskiy, V., Banaru, M., Hallen, L., Krobitsch, S., Lehrach, H., Soldatov, A. (2009). Transcriptome analysis by strand-specific sequencing of complementary DNA. Nucleic Acids Res.37(18), e123], which has been recently reviewed as the leading protocol for strand-specific RNA-Seq library preparation [Levin, J. Z., Yassour, M., Adiconis, X., Nusbaum, C., Thompson, D. A., Friedman, N., Gnirke, A., Regev, A. (2009). Comprehensive comparative analysis of strand-specific RNA sequencing methods. Nat. Methods7(9), 709-715]. The procedure starts with the isolation of the polyA fraction (mRNA) within a pool of total RNA, followed by its fragmentation. Then double-stranded (ds) cDNA synthesis is performed with the incorporation of dUTP in the second strand. The ds cDNA fragments are further processed following a standard sequencing library preparation scheme tailored for the Illumina sequencing platform: end polishing, A-tailing, adapter ligation, and size selection. Prior to final amplification, the dUTP-marked strand is selectively degraded by Uracil-DNA-Glycosylase (UDG). The remaining strand is amplified to generate a cDNA library suitable for sequencing.
