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Nucleic Acids Res. 2011 Oct;39(18):e120. doi: 10.1093/nar/gkr547. Epub 2011 Jul 6.

Whole-transcriptome RNAseq analysis from minute amount of total RNA.

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  • 1Department of Biomolecular Engineering, University of California Santa Cruz, 1156 High Street, Santa Cruz, CA 95064, USA.

Abstract

RNA sequencing approaches to transcriptome analysis require a large amount of input total RNA to yield sufficient mRNA using either poly-A selection or depletion of rRNA. This feature makes it difficult to miniaturize transcriptome analysis for greater efficiency. To address this challenge, we devised and validated a simple procedure for the preparation of whole-transcriptome cDNA libraries from a minute amount (500 pg) of total RNA. We compared a single-sample library prepared by this Ovation RNA-Seq system with two available methods of mRNA enrichment (TruSeq poly-A enrichment and RiboMinus rRNA depletion). Using the Ovation preparation method for a set of eight mouse tissue samples, the RNA sequencing data obtained from two different next-generation sequencing platforms (SOLiD and Illumina Genome Analyzer IIx) yielded negligible rRNA reads (<3.5%) while retaining transcriptome sequencing fidelity. We further validated the Ovation amplification technique by examining the resulting library complexity, reproducibility, evenness of transcript coverage, 5' and 3' bias and platform-specific biases. Notably, in this side-by-side comparison, SOLiD sequencing chemistry is biased toward higher GC content of transcriptome and Illumina Genome analyzer IIx is biased away from neutral to lower GC content of the transcriptomics regions.

PMID:
21737426
[PubMed - indexed for MEDLINE]
PMCID:
PMC3185437
Free PMC Article

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