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Increasing quality, throughput and speed of sample preparation for strand-specific messenger RNA sequencing

2017; BioMed Central; Volume: 18; Issue: 1 Linguagem: Inglês

10.1186/s12864-017-3900-6

1471-2164

Simon Haile, Richard Corbett, Tina MacLeod, Steve Bilobram, Duane E. Smailus, Philip S. Tsao, Heather Kirk, Helen McDonald, Pawan Pandoh, Miruna Bala, Martin Hirst, Diane Miller, Richard A. Moore, Andrew J. Mungall, Jacquie Schein, Robin Coope, Yussanne Ma, Yongjun Zhao, Robert A. Holt, Steven J.M. Jones, Marco A. Marra,

RNA and protein synthesis mechanisms

RNA-Sequencing (RNA-seq) is now commonly used to reveal quantitative spatiotemporal snapshots of the transcriptome, the structures of transcripts (splice variants and fusions) and landscapes of expressed mutations. However, standard approaches for library construction typically require relatively high amounts of input RNA, are labor intensive, and are time consuming. Here, we report the outcome of a systematic effort to optimize and streamline steps in strand-specific RNA-seq library construction. This work has resulted in the identification of an optimized messenger RNA isolation protocol, a potent reverse transcriptase for cDNA synthesis, and an efficient chemistry and a simplified formulation of library construction reagents. We also present an optimization of bead-based purification and size selection designed to maximize the recovery of cDNA fragments. These developments have allowed us to assemble a rapid high throughput pipeline that produces high quality data from amounts of total RNA as low as 25 ng. While the focus of this study is on RNA-seq sample preparation, some of these developments are also relevant to other next-generation sequencing library types.