Ribosomal RNA Depletion for Efficient Use of RNA-Seq Capacity

A decade since its invention, single-cell RNA sequencing (scRNA-seq) has become a mainstay technology for profiling transcriptional heterogeneity in individual cells. Yet, most existing scRNA-seq methods capture only polyadenylated mRNA to avoid the cost of sequencing non-messenger transcripts, such as ribosomal RNA (rRNA), that are usually not of-interest. Hence, there are not very many protocols that enable single-cell analysis of total RNA. We adapted a method called DASH (Depletion of Abundant Sequences by Hybridisation) to make it suitable for depleting rRNA sequences from single-cell total RNA-seq libraries. Our analyses show that our single-cell DASH (scDASH) method can effectively deplete rRNAs from sequencing libraries with minimal off-target non-specificity. Importantly, as a result of depleting the rRNA, the rest of the transcriptome is significantly enriched for detection.

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Paired rRNA-depleted and polyA-selected RNA sequencing data and supporting multi-omics data from human T cells

Scientific Data ◽

10.1038/s41597-020-00719-4 ◽

2020 ◽

Vol 7 (1) ◽

Author(s):

Li Chen ◽

Ruirui Yang ◽

Tony Kwan ◽

Chao Tang ◽

Stephen Watt ◽

...

Keyword(s):

Rna Sequencing ◽

Ribosomal Rna ◽

Rna Seq ◽

Healthy Individuals ◽

Sequencing Data ◽

Advantages And Disadvantages ◽

Human Primary Cells ◽

Human T Cells ◽

Differential Gene ◽

Rna Depletion

Abstract Both poly(A) enrichment and ribosomal RNA depletion are commonly used for RNA sequencing. Either has its advantages and disadvantages that may lead to biases in the downstream analyses. To better access these effects, we carried out both ribosomal RNA-depleted and poly(A)-selected RNA-seq for CD4+ T naive cells isolated from 40 healthy individuals from the Blueprint Project. For these 40 individuals, the genomic and epigenetic data were also available. This dataset offers a unique opportunity to understand how library construction influences differential gene expression, alternative splicing and molecular QTL (quantitative loci) analyses for human primary cells.

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An Easy, Cost‐Effective, and Scalable Method to Deplete Human Ribosomal RNA for RNA‐seq

Current Protocols ◽

10.1002/cpz1.176 ◽

2021 ◽

Vol 1 (6) ◽

Author(s):

Amber Baldwin ◽

Adam R. Morris ◽

Neelanjan Mukherjee

Keyword(s):

Ribosomal Rna ◽

Cost Effective ◽

Rna Seq

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Dual RNA-seq analysis of in vitro infection multiplicity and RNA depletion methods in Chlamydia-infected epithelial cells

Scientific Reports ◽

10.1038/s41598-021-89921-x ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Regan J. Hayward ◽

Michael S. Humphrys ◽

Wilhelmina M. Huston ◽

Garry S. A. Myers

Keyword(s):

Epithelial Cells ◽

Multiplicity Of Infection ◽

Rna Seq ◽

Infection Rates ◽

Time Points ◽

High Infection ◽

Infection Cycles ◽

Transcriptomic Responses ◽

Rna Depletion

AbstractDual RNA-seq experiments examining viral and bacterial pathogens are increasing, but vary considerably in their experimental designs, such as infection rates and RNA depletion methods. Here, we have applied dual RNA-seq to Chlamydia trachomatis infected epithelial cells to examine transcriptomic responses from both organisms. We compared two time points post infection (1 and 24 h), three multiplicity of infection (MOI) ratios (0.1, 1 and 10) and two RNA depletion methods (rRNA and polyA). Capture of bacterial-specific RNA were greatest when combining rRNA and polyA depletion, and when using a higher MOI. However, under these conditions, host RNA capture was negatively impacted. Although it is tempting to use high infection rates, the implications on host cell survival, the potential reduced length of infection cycles and real world applicability should be considered. This data highlights the delicate nature of balancing host–pathogen RNA capture and will assist future transcriptomic-based studies to achieve more specific and relevant infection-related biological insights.

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Biogenesis and Function of Small Non-Coding RNAs Derived from Eukaryotic Ribosomal RNA

10.20944/preprints201810.0596.v1 ◽

2018 ◽

Author(s):

Marine Lambert ◽

Abderrahim Benmoussa ◽

Patrick Provost

Keyword(s):

Ribosomal Rna ◽

Potential Role ◽

A Priori ◽

Biological Significance ◽

Rna Seq ◽

New Class ◽

Scientific Mind ◽

Non Coding Rnas ◽

And Function ◽

Rrna Sequences

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

Nature Methods ◽

10.1038/nmeth.f.352 ◽

2011 ◽

Vol 8 (11) ◽

pp. iii-iv ◽

Cited By ~ 9

Author(s):

Vladimir Benes ◽

Jonathon Blake ◽

Ken Doyle

Keyword(s):

Ribosomal Rna ◽

Rna Seq

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Ribo-Pop: simple, cost-effective, and widely applicable ribosomal RNA depletion

RNA ◽

10.1261/rna.076562.120 ◽

2020 ◽

Vol 26 (11) ◽

pp. 1731-1742 ◽

Cited By ~ 1

Author(s):

Mary Kay Thompson ◽

Maria Kiourlappou ◽

Ilan Davis

Keyword(s):

Ribosomal Rna ◽

Cost Effective ◽

Rna Depletion

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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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