Selective Depletion of rRNA Enables Whole Transcriptome Profiling of Archival Fixed Tissue

Morlan, John; Qu, Kunbin; Sinicropi, Dominick

doi:10.1371/journal.pone.0042882

Cited by 189 publications

(178 citation statements)

References 13 publications

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“…A wide range of variables was evaluated, including library preparation methods (polyA-enriched and ribo-depleted), size-specific fractionation (1, 2 and 3 kb) and RNA integrity (using heat, RNase A and sonication to degrade the RNA). The latter variable was chosen to mimic some of the damaging effects of tissue fixation with formalin, which is a well-recognized issue for RNA profiling of formalin-fixed, paraffin-embedded (FFPE) clinical specimens 22-24 . Finally, we leveraged a data set of 18,124 PrimePCR reactions and used it with 802 previously published 10 TaqMan RT-qPCR reactions as orthogonal measurements to gauge the linear response and dynamic range of the RNA-seq results from the different platforms and protocols.…”

Section: Introductionmentioning

confidence: 99%

Multi-platform assessment of transcriptome profiling using RNA-seq in the ABRF next-generation sequencing study

et al. 2014

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High-throughput RNA sequencing (RNA-seq) dramatically expands the potential for novel genomics discoveries, but the wide variety of platforms, protocols and performance has created the need for comprehensive reference data. Here we describe the Association of Biomolecular Resource Facilities next-generation sequencing (ABRF-NGS) study on RNA-seq. We tested replicate experiments across 15 laboratory sites using reference RNA standards to test four protocols (polyA-selected, ribo-depleted, size-selected and degraded) on five sequencing platforms (Illumina HiSeq, Life Technologies’ PGM and Proton, Pacific Biosciences RS and Roche’s 454). The results show high intra-platform and inter-platform concordance for expression measures across the deep-count platforms, but highly variable efficiency and cost for splice junction and variant detection between all platforms. These data also demonstrate that ribosomal RNA depletion can both enable effective analysis of degraded RNA samples and be readily compared to polyA-enriched fractions. This study provides a broad foundation for cross-platform standardization, evaluation and improvement of RNA-seq.

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Section: Introductionmentioning

confidence: 99%

Multi-platform assessment of transcriptome profiling using RNA-seq in the ABRF next-generation sequencing study

et al. 2014

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“…To alleviate the detrimental effects of poly(rA) RNA carrier on sequencing quality, we developed a targeted RNase-H-based depletion method [25] to remove it prior to library construction. We used 40mer oligo(dT) probes to form RNase H-cleavable DNA-RNA hybrids with poly(rA) (Figure 1C), which successfully depleted poly(rA) from a sample with carrier added (Figure 1A; right panel).…”

Section: Resultsmentioning

confidence: 99%

Enhanced methods for unbiased deep sequencing of Lassa and Ebola RNA viruses from clinical and biological samples

et al. 2014

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We have developed a robust RNA sequencing method for generating complete de novo assemblies with intra-host variant calls of Lassa and Ebola virus genomes in clinical and biological samples. Our method uses targeted RNase H-based digestion to remove contaminating poly(rA) carrier and ribosomal RNA. This depletion step improves both the quality of data and quantity of informative reads in unbiased total RNA sequencing libraries. We have also developed a hybrid-selection protocol to further enrich the viral content of sequencing libraries. These protocols have enabled rapid deep sequencing of both Lassa and Ebola virus and are broadly applicable to other viral genomics studies.

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“…~60 L2–L4 mixed gender animals were picked and flash frozen in 50mM NaCl, and RNA extracted with trizol. RNaseH and 94 oligos complementary to ribosomal RNA were used to deplete rRNA from the sample 48 . Briefly, ~250ng of a cocktail of DNA oligos complementary to rRNA (Supplementary Table 3, ordered from Integrated DNA Technologies) was mixed with ~100ng total RNA in 125mM Tris pH7.4, 250mM NaCl in 8ul.…”

Section: Methodsmentioning

confidence: 99%

Translation readthrough mitigation

Arribere

Cenik

Jain

et al. 2016

Nature

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A fraction of ribosomes engaged in translation will fail to terminate when reaching a stop codon, yielding nascent proteins inappropriately extended on their C-termini. Although such extended proteins can interfere with normal cellular processes, known mechanisms of translational surveillance are insufficient to protect cells from potential dominant consequences. Through a combination of transgenics and CRISPR/Cas9 gene editing in C. elegans, we demonstrate a consistent ability of cells to block accumulation of C-terminal extended proteins that result from failure to terminate at stop codons. 3’UTR-encoded sequences were sufficient to lower protein levels. Measurements of mRNA levels and translation suggested a co- or post-translational mechanism of action for these sequences in C. elegans. Similar mechanisms evidently operate in human cells, where we observed a comparable tendency for translated human 3’UTR sequences to reduce mature protein expression in tissue culture assays, including 3' sequences from the hypomorphic “Constant Spring” hemoglobin stop codon variant. We suggest 3’UTRs may encode peptide sequences that destabilize the attached protein, providing mitigation of unwelcome and varied translation errors.

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Selective Depletion of rRNA Enables Whole Transcriptome Profiling of Archival Fixed Tissue

Cited by 189 publications

References 13 publications

Multi-platform assessment of transcriptome profiling using RNA-seq in the ABRF next-generation sequencing study

Multi-platform assessment of transcriptome profiling using RNA-seq in the ABRF next-generation sequencing study

Enhanced methods for unbiased deep sequencing of Lassa and Ebola RNA viruses from clinical and biological samples

Translation readthrough mitigation

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