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A simple and novel method for RNA-seq library preparation of single cell cDNA analysis by hyperactive Tn5 transposase.
Dev Dyn. 2012 Aug 21;
Authors: Brouilette S, Kuersten S, Mein C, Bozek M, Terry A, Dias KR, Bhaw-Rosun L, Shintani Y, Coppen S, Ikebe C, Sawhney V, Campbell N, Kaneko M, Tano N, Ishida H, Suzuki K, Yashiro K
Abstract
BACKGROUND: Deep sequencing of single cell-derived cDNAs offers novel insights into oncogenesis and embryogenesis. However, traditional library preparation for RNA-seq analysis requires multiple steps with consequent sample loss and stochastic variation at each step significantly affecting output. Thus a simpler and better protocol is desirable. The recently developed hyperactive Tn5-mediated library preparation, which brings high quality libraries, is likely one of the solutions. RESULTS AND CONCLUSIONS: Here we tested the applicability of hyperactive Tn5-mediated library preparation to deep sequencing of single cell cDNA, optimized the protocol, and compared it with the conventional method based on sonication. This new technique does not require any expensive or special equipment, which secures wider availability. A library was constructed from only 100 ng of cDNA, which enables the saving of precious specimens. Only a few steps of robust enzymatic reaction resulted in saved time, enabling more specimens to be prepared at once, and with a more reproducible size distribution among the different specimens. The obtained RNA-seq results were comparable to the conventional method. Thus this Tn5-mediated preparation is applicable for anyone who aims to carry out deep sequencing for single cell cDNAs.
PMID: 22911638 [PubMed - as supplied by publisher]
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A simple and novel method for RNA-seq library preparation of single cell cDNA analysis by hyperactive Tn5 transposase.
Dev Dyn. 2012 Aug 21;
Authors: Brouilette S, Kuersten S, Mein C, Bozek M, Terry A, Dias KR, Bhaw-Rosun L, Shintani Y, Coppen S, Ikebe C, Sawhney V, Campbell N, Kaneko M, Tano N, Ishida H, Suzuki K, Yashiro K
Abstract
BACKGROUND: Deep sequencing of single cell-derived cDNAs offers novel insights into oncogenesis and embryogenesis. However, traditional library preparation for RNA-seq analysis requires multiple steps with consequent sample loss and stochastic variation at each step significantly affecting output. Thus a simpler and better protocol is desirable. The recently developed hyperactive Tn5-mediated library preparation, which brings high quality libraries, is likely one of the solutions. RESULTS AND CONCLUSIONS: Here we tested the applicability of hyperactive Tn5-mediated library preparation to deep sequencing of single cell cDNA, optimized the protocol, and compared it with the conventional method based on sonication. This new technique does not require any expensive or special equipment, which secures wider availability. A library was constructed from only 100 ng of cDNA, which enables the saving of precious specimens. Only a few steps of robust enzymatic reaction resulted in saved time, enabling more specimens to be prepared at once, and with a more reproducible size distribution among the different specimens. The obtained RNA-seq results were comparable to the conventional method. Thus this Tn5-mediated preparation is applicable for anyone who aims to carry out deep sequencing for single cell cDNAs.
PMID: 22911638 [PubMed - as supplied by publisher]
More...