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BMC Genomics

, 18:515

Transcriptomic methods

Abstract

BackgroundRNA-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.

MethodsHere, we report the outcome of a systematic effort to optimize and streamline steps in strand-specific RNA-seq library construction.

ResultsThis 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.

ConclusionsThese 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.

KeywordsAmpure XP magnetic beads Next-generation sequencing Library construction Strand-specific dUTP Uracil DNA N-Glycosylase Ligation mRNA RNA-seq Illumina Ligation Reverse transcriptase AbbreviationsERCCExternal RNA Controls Consortium

mRNAMessenger RNA

NGSNext Generation Sequencing

RNA-SeqRNA sequencing

rRNARibosomal RNA

RTReverse Transcriptase

SSIISuperscript II

ssRNA-seqStrand-specific RNA-seq

Electronic supplementary materialThe online version of this article doi:10.1186-s12864-017-3900-6 contains supplementary material, which is available to authorized users.

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Autor: Simon Haile - Richard D. Corbett - Tina MacLeod - Steve Bilobram - Duane Smailus - Philip Tsao - Heather Kirk - Helen McD

Fuente: https://link.springer.com/



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