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

Authors: Simon Haile; Richard Corbett; Tina MacLeod; Steve Bilobram; Duane E Smailus; Philip Tsao; Heather Kirk; +14 Authors

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

Abstract

Background 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. Methods Here, we report the outcome of a systematic effort to optimize and streamline steps in strand-specific RNA-seq library construction. Results 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. Conclusions 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.

Subjects by Vocabulary

Library of Congress Subject Headings: lcsh:QH426-470 lcsh:Biotechnology lcsh:TP248.13-248.65 lcsh:Genetics

Microsoft Academic Graph classification: RNA-Seq Computational biology Biology DNA sequencing Transcriptome Complementary DNA Genetics Messenger RNA RNA Reverse transcriptase DNA microarray

Keywords

Library construction, mRNA, Strand-specific, Ampure XP magnetic beads, HL-60 Cells, Specimen Handling, Illumina, Reverse transcriptase, Genetics, Humans, RNA, Messenger, Ligation, Gene Library, Sequence Analysis, RNA, Methodology Article, Uracil DNA N-Glycosylase, dUTP, Next-generation sequencing, RNA-seq, Biotechnology

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  • citations
    This is an alternative to the "Influence" indicator, which also reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
    17
    popularity
    This indicator reflects the "current" impact/attention (the "hype") of an article in the research community at large, based on the underlying citation network.
    Top 10%
    influence
    This indicator reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
    Average
    impulse
    This indicator reflects the initial momentum of an article directly after its publication, based on the underlying citation network.
    Top 10%
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citations
This is an alternative to the "Influence" indicator, which also reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
BIP!Citations provided by BIP!
popularity
This indicator reflects the "current" impact/attention (the "hype") of an article in the research community at large, based on the underlying citation network.
BIP!Popularity provided by BIP!
influence
This indicator reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
BIP!Influence provided by BIP!
impulse
This indicator reflects the initial momentum of an article directly after its publication, based on the underlying citation network.
BIP!Impulse provided by BIP!
17
Top 10%
Average
Top 10%
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