title: cDNA normalization by hydroxyapatite chromatography to enrich transcriptome diversity in RNA-seq applications.
abstract: 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 (HAC) 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 HAC-based normalization enriched for all non-ribosomal RNA species regardless of RNA transcript abundance or length when compared with untreated controls. Microcolumn HAC 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 HAC as an effective, cost saving, and non-destructive alternative normalization technique.
authors.affiliation: Biotechnology and Bioengineering Department, Sandia National Laboratories, Livermore, CA, USA.
authors.forename: Victoria A
authors.initials: VA
authors.lastname: Vandernoot
authors.forename: Stanley A
authors.initials: SA
authors.lastname: Langevin
authors.forename: Owen D
authors.initials: OD
authors.lastname: Solberg
authors.forename: Pamela D
authors.initials: PD
authors.lastname: Lane
authors.forename: Deanna J
authors.initials: DJ
authors.lastname: Curtis
authors.forename: Zachary W
authors.initials: ZW
authors.lastname: Bent
authors.forename: Kelly P
authors.initials: KP
authors.lastname: Williams
authors.forename: Kamlesh D
authors.initials: KD
authors.lastname: Patel
authors.forename: Joseph S
authors.initials: JS
authors.lastname: Schoeniger
authors.forename: Steven S
authors.initials: SS
authors.lastname: Branda
authors.forename: Todd W
authors.initials: TW
authors.lastname: Lane
mesh_terms: D001483:Base Sequence; D002846:Chromatography, Affinity / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods; D002874:Chromosome Mapping; D017886:Durapatite / Q000737:chemistry*; D048168:Escherichia coli K12 / Q000235:genetics; D015723:Gene Library; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000737:chemistry; D012313:RNA / Q000032:analysis / Q000737:chemistry / Q000235:genetics*; D017423:Sequence Analysis, RNA / Q000379:methods*; D059467:Transcriptome
chemical_list: D012313:RNA; D017886:Durapatite