期刊
BIOTECHNIQUES
卷 53, 期 6, 页码 373-+出版社
FUTURE SCI LTD
DOI: 10.2144/000113937
关键词
hydroxyapatite chromatography; nucleic acid normalization; microcolumn; rRNA depletion; RNA-seq; second generation sequencing; C(0)t filtration
资金
- U.S. Department of Energy's National Nuclear Security Administration [DE-AC04-94AL85000]
Second-generation sequencing (SGS) has become the preferred method for RNA transcriptome profiling of organisms and single cells. However, SGS analysis of transcriptome diversity (including protein-coding transcripts and regulatory non-coding RNAs) is inefficient unless the sample of interest is first depleted of nucleic acids derived from ribosomal RNA (rRNA), which typically account for up to 95% of total intracellular RNA content. Here we describe a novel microscale hydroxyapatite chromatography (HAG) normalization method to remove eukaryotic and prokaryotic high abundant rRNA species, thereby increasing sequence coverage depth and transcript diversity across non-rRNA populations. RNA-seq analysis of Escherichia coli K-12 and human intracellular total RNA showed that HAG-based normalization enriched for all non-ribosomal RNA species regardless of RNA transcript abundance or length when compared with untreated controls. Microcolumn HAG normalization generated rRNA-depleted cDNA libraries comparable to the well-established duplex specific nuclease (DSN) normalization and Ribo-Zero rRNA-depletion methods, thus establishing microscale HAG as an effective, cost saving, and non-destructive alternative normalization technique.
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