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A simple and novel method for RNA‐seq library preparation of single cell cDNA analysis by hyperactive Tn5 transposase
Author(s) -
Brouilette Scott,
Kuersten Scott,
Mein Charles,
Bozek Monika,
Terry Anna,
Dias KerithRae,
BhawRosun Leena,
Shintani Yasunori,
Coppen Steven,
Ikebe Chiho,
Sawhney Vinit,
Campbell Niall,
Kaneko Masahiro,
Tano Nobuko,
Ishida Hidekazu,
Suzuki Ken,
Yashiro Kenta
Publication year - 2012
Publication title -
developmental dynamics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.634
H-Index - 141
eISSN - 1097-0177
pISSN - 1058-8388
DOI - 10.1002/dvdy.23850
Subject(s) - biology , cdna library , complementary dna , computational biology , genomic library , single cell sequencing , rna , rna seq , deep sequencing , computer science , genetics , microbiology and biotechnology , gene , transcriptome , genome , base sequence , phenotype , gene expression , exome sequencing
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. Developmental Dynamics 241:1584–1590, 2012. © 2012 Wiley Periodicals Inc.