Ribo-Zero Gold Kit: improved RNA-seq results after removal of cytoplasmic and mitochondrial ribosomal RNA

The advent of RNA-sequencing (RNA-Seq) technologies has markedly improved our knowledge and expanded the compendium of small non-coding RNAs, most of which derive from the processing of longer RNA precursors. In this review article, we will discuss about the biogenesis and function of small non-coding RNAs derived from eukaryotic ribosomal RNA (rRNA), called rRNA fragments (rRFs), and their potential role(s) as regulator of gene expression. This relatively new class of ncRNAs remained poorly investigated and underappreciated until recently, due mainly to the a priori exclusion of rRNA sequences—because of their overabundance—from RNA-Seq datasets. The situation surrounding rRFs resembles that of microRNAs (miRNAs), which used to be readily discarded from further analyses, for more than five decades, because we could not believe that RNA of such a short length could bear biological significance. As if we had not yet learned our lesson not to restrain our investigative, scientific mind from challenging widely accepted beliefs or dogmas, and from looking for the hidden treasures in the most unexpected places.

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Comparison of Poly-A+ Selection and rRNA Depletion in Detection of lncRNA in Two Equine Tissues Using RNA-seq

Non-Coding RNA ◽

10.3390/ncrna6030032 ◽

2020 ◽

Vol 6 (3) ◽

pp. 32 ◽

Cited By ~ 1

Author(s):

Anna R. Dahlgren ◽

Erica Y. Scott ◽

Tamer Mansour ◽

Erin N. Hales ◽

Pablo J. Ross ◽

...

Keyword(s):

Ribosomal Rna ◽

Preparation Method ◽

Parietal Lobe ◽

Library Preparation ◽

Rna Seq ◽

The Past ◽

Rrna Depletion ◽

Non Coding Rnas ◽

Nucleolar Rna ◽

Library Preparation Method

Long non-coding RNAs (lncRNAs) are untranslated regulatory transcripts longer than 200 nucleotides that can play a role in transcriptional, post-translational, and epigenetic regulation. Traditionally, RNA-sequencing (RNA-seq) libraries have been created by isolating transcriptomic RNA via poly-A+ selection. In the past 10 years, methods to perform ribosomal RNA (rRNA) depletion of total RNA have been developed as an alternative, aiming for better coverage of whole transcriptomic RNA, both polyadenylated and non-polyadenylated transcripts. The purpose of this study was to determine which library preparation method is optimal for lncRNA investigations in the horse. Using liver and cerebral parietal lobe tissues from two healthy Thoroughbred mares, RNA-seq libraries were prepared using standard poly-A+ selection and rRNA-depletion methods. Averaging the two biologic replicates, poly-A+ selection yielded 327 and 773 more unique lncRNA transcripts for liver and parietal lobe, respectively. More lncRNA were found to be unique to poly-A+ selected libraries, and rRNA-depletion identified small nucleolar RNA (snoRNA) to have a higher relative expression than in the poly-A+ selected libraries. Overall, poly-A+ selection provides a more thorough identification of total lncRNA in equine tissues while rRNA-depletion may allow for easier detection of snoRNAs.

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A resource of ribosomal RNA-depleted RNA-Seq data from different normal adult and fetal human tissues

Scientific Data ◽

10.1038/sdata.2015.63 ◽

2015 ◽

Vol 2 (1) ◽

Cited By ~ 13

Author(s):

Jocelyn Y.H. Choy ◽

Priscilla L.S. Boon ◽

Nicolas Bertin ◽

Melissa J. Fullwood

Keyword(s):

Ribosomal Rna ◽

Normal Adult ◽

Human Tissues ◽

Rna Seq

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A CLEAR pipeline for direct comparison of circular and linear RNA expression

10.1101/668657 ◽

2019 ◽

Author(s):

Xu-Kai Ma ◽

Meng-Ran Wang ◽

Chu-Xiao Liu ◽

Rui Dong ◽

Gordon G. Carmichael ◽

...

Keyword(s):

Ribosomal Rna ◽

Circular Rnas ◽

Rna Expression ◽

Computational Pipeline ◽

Rna Seq ◽

Link Type ◽

Genome Wide ◽

A Genome

ABSTRACTSequences of circular RNAs (circRNAs) produced from back-splicing of exon(s) completely overlap with sequences from cognate linear RNAs transcribed from the same gene loci with the exception of their back-splicing junction (BSJ) sites. Examination of global circRNA expression from RNA-seq datasets generally relies on the detection of RNA-seq fragments spanning BSJ sites, but a direct comparison of circular and linear RNA expression from the same gene loci in a genome-wide manner has remained challenging. This is because quantification of BSJ fragments differs from that of linear RNA expression that uses normalized RNA-seq fragments mapped to the whole gene bodies. Here, we have developed a computational pipeline for circular and linear RNA expression analysis from ribosomal-RNA depleted RNA-seq (CLEAR, https://github.com/YangLab/CLEAR). A new quantitation parameter, FPB (fragments per billion mapped bases), is applied to evaluate circular and linear RNA expression individually by fragments mapped to circRNA-specific BSJ sites or to linear RNA-specific splicing junction (SJ) sites. Then, circular and linear RNA expression are directly compared by dividing FPBcirc by FPBlinear to generate a CIRCscore, which indicates the relative circRNA expression using linear RNA expression as the background. Highly-expressed circRNAs with low cognate linear RNA expression background can be identified for further investigation.

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Incomplete removal of ribosomal RNA can affect chromatin RNA-seq data analysis

Transcription ◽

10.1080/21541264.2020.1794491 ◽

2020 ◽

Vol 11 (5) ◽

pp. 230-235

Author(s):

Michael Tellier ◽

Shona Murphy

Keyword(s):

Data Analysis ◽

Ribosomal Rna ◽

Rna Seq ◽

Incomplete Removal

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Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq

Journal of Visualized Experiments ◽

10.3791/50093 ◽

2013 ◽

Cited By ~ 8

Author(s):

Phanidhar Kukutla ◽

Matthew Steritz ◽

Jiannong Xu

Keyword(s):

Ribosomal Rna ◽

Rna Seq

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Efficient depletion of ribosomal RNA for RNA sequencing in planarians

10.1101/670604 ◽

2019 ◽

Author(s):

Iana V. Kim ◽

Eric J. Ross ◽

Sascha Dietrich ◽

Kristina Döring ◽

Alejandro Sánchez Alvarado ◽

...

Keyword(s):

Stem Cells ◽

Ribosomal Rna ◽

Cost Effective ◽

Subtractive Hybridization ◽

Mrna Levels ◽

Rna Seq ◽

Planarian Regeneration ◽

Histone Mrnas ◽

Transposon Activity ◽

The Impact

AbstractBackgroundThe astounding regenerative abilities of planarian flatworms prompt a steadily growing interest in examining their molecular foundation. Planarian regeneration was found to require hundreds of genes and is hence a complex process. Thus, RNA interference followed by transcriptome-wide gene expression analysis by RNA-seq is a popular technique to study the impact of any particular planarian gene on regeneration. Typically, the removal of ribosomal RNA (rRNA) is the first step of all RNA-Seq library preparation protocols. To date, rRNA removal in planarians was primarily achieved by the enrichment of polyadenylated (poly(A)) transcripts. However, to better reflect transcriptome dynamics and to cover also non-poly(A) transcripts, a procedure for the targeted removal of rRNA in planarians is needed.ResultsIn this study, we describe a workflow for the efficient depletion of rRNA in the planarian model species S. mediterranea. Our protocol is based on subtractive hybridization using organism-specific probes. Importantly, the designed probes also deplete rRNA of other freshwater triclad families, a fact that considerably broadens the applicability of our protocol. We tested our approach on total RNA isolated stem cells (termed neoblasts) of S. mediterranea and compared ribodepleted libraries with publicly available poly(A)-enriched ones. Overall, mRNA levels after ribodepletion were consisted with poly(A) libraries. However, ribodepleted libraries revealed higher transcript levels for transposable elements and histone mRNAs that remained underrepresented in poly(A) libraries. As neoblasts experience high transposon activity this suggests that ribodepleted libraries better reflect the transcriptional dynamics of planarian stem cells. Furthermore, the presented ribodepletion procedure was successfully expanded to the removal of ribosomal RNA from the gram-negative bacterium Salmonella typhimurium.ConclusionsThe ribodepletion protocol presented here ensures the efficient rRNA removal from low input total planarian RNA, which can be further processed for RNA-Seq applications. Resulting libraries contain less than 2% rRNA. Moreover, for a cost-effective and efficient removal of rRNA prior to sequencing applications our procedure might be adapted to any prokaryotic or eukaryotic species of choice.

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Multiple freeze-thaw cycles lead to a loss of consistency in poly(A)-enriched RNA sequencing

10.21203/rs.3.rs-67621/v2 ◽

2020 ◽

Author(s):

Benjamin Kellman ◽

Hratch Baghdassarian ◽

Tiziano Pramparo ◽

Isaac Shamie ◽

Vahid Gazestani ◽

...

Keyword(s):

Differential Expression ◽

Rna Sequencing ◽

Ribosomal Rna ◽

Rna Degradation ◽

Library Preparation ◽

Rna Seq ◽

Rna Integrity ◽

Freeze Thaw ◽

The Impact ◽

Do So

Abstract Background: Both RNA-Seq and sample freeze-thaw are ubiquitous. However, knowledge about the impact of freeze-thaw on downstream analyses is limited. The lack of common quality metrics that are sufficiently sensitive to freeze-thaw and RNA degradation, e.g. the RNA Integrity Score, makes such assessments challenging.Results: Here we quantify the impact of repeated freeze-thaw cycles on the reliability of RNA-Seq by examining poly(A)-enriched and ribosomal RNA depleted RNA-seq from frozen leukocytes drawn from a toddler Autism cohort. To do so, we estimate the relative noise, or percentage of random counts, separating technical replicates. Using this approach we measured noise associated with RIN and freeze-thaw cycles. As expected, RIN does not fully capture sample degradation due to freeze-thaw. We further examined differential expression results and found that three freeze-thaws should extinguish the differential expression reproducibility of similar experiments. Freeze-thaw also resulted in a 3’ shift in the read coverage distribution along the gene body of poly(A)-enriched samples compared to ribosomal RNA depleted samples, suggesting that library preparation may exacerbate freeze-thaw-induced sample degradation.Conclusion: The use of poly(A)-enrichment for RNA sequencing is pervasive in library preparation of frozen tissue, and thus, it is important during experimental design and data analysis to consider the impact of repeated freeze-thaw cycles on reproducibility.

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