Strand-specific libraries for high throughput RNA sequencing (RNA-Seq) prepared without poly(A) selection
UMass Chan Affiliations
Program in Molecular MedicineProgram in Bioinformatics and Integrative Biology
Department of Biochemistry and Molecular Pharmacology
Document Type
Journal ArticlePublication Date
2012-12-28Keywords
High-Throughput Nucleotide SequencingSequence Analysis, RNA
Bioinformatics
Computational Biology
Genetics and Genomics
Molecular Biology
Systems Biology
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Show full item recordAbstract
BACKGROUND: High throughput DNA sequencing technology has enabled quantification of all the RNAs in a cell or tissue, a method widely known as RNA sequencing (RNA-Seq). However, non-coding RNAs such as rRNA are highly abundant and can consume >70% of sequencing reads. A common approach is to extract only polyadenylated mRNA; however, such approaches are blind to RNAs with short or no poly(A) tails, leading to an incomplete view of the transcriptome. Another challenge of preparing RNA-Seq libraries is to preserve the strand information of the RNAs. DESIGN: Here, we describe a procedure for preparing RNA-Seq libraries from 1 to 4 mug total RNA without poly(A) selection. Our method combines the deoxyuridine triphosphate (dUTP)/uracil-DNA glycosylase (UDG) strategy to achieve strand specificity with AMPure XP magnetic beads to perform size selection. Together, these steps eliminate gel purification, allowing a library to be made in less than two days. We barcode each library during the final PCR amplification step, allowing several samples to be sequenced in a single lane without sacrificing read length. Libraries prepared using this protocol are compatible with Illumina GAII, GAIIx and HiSeq 2000 platforms. DISCUSSION: The RNA-Seq protocol described here yields strand-specific transcriptome libraries without poly(A) selection, which provide approximately 90% mappable sequences. Typically, more than 85% of mapped reads correspond to protein-coding genes and only 6% derive from non-coding RNAs. The protocol has been used to measure RNA transcript identity and abundance in tissues from flies, mice, rats, chickens, and frogs, demonstrating its general applicability.Source
Silence. 2012 Dec 28;3(1):9. doi: 10.1186/1758-907X-3-9. Link to article on publisher's siteDOI
10.1186/1758-907X-3-9Permanent Link to this Item
http://hdl.handle.net/20.500.14038/25888PubMed ID
23273270Notes
Author Zhao Zhang is a student in the Interdisciplinary Graduate Program in the Graduate School of Biomedical Sciences (GSBS) at UMass Medical School.
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Link to Article in PubMedRights
© 2012 Zhang et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.ae974a485f413a2113503eed53cd6c53
10.1186/1758-907X-3-9