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

2021 ◽  
Vol 1 (6) ◽  
Author(s):  
Amber Baldwin ◽  
Adam R. Morris ◽  
Neelanjan Mukherjee
Keyword(s):  
Ribosomal Rna ◽  
Cost Effective ◽  
Rna Seq

scholarly journals Efficient depletion of ribosomal RNA for RNA sequencing in planarians

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.

scholarly journals A simple, cost-effective, and robust method for rRNA depletion in RNA-sequencing studies

2020 ◽  
Author(s):  
Peter H. Culviner ◽  
Chantal K. Guegler ◽  
Michael T. Laub
Keyword(s):  
Gene Expression ◽  
Rna Sequencing ◽  
Ribosomal Rna ◽  
Bacterial Species ◽  
Cost Effective ◽  
Rna Seq ◽  
Robust Method ◽  
Total Rna ◽  
Metagenomic Sample ◽  
Rrna Depletion

AbstractThe profiling of gene expression by RNA-sequencing (RNA-seq) has enabled powerful studies of global transcriptional patterns in all organisms, including bacteria. Because the vast majority of RNA in bacteria is ribosomal RNA (rRNA), it is standard practice to deplete the rRNA from a total RNA sample such that the reads in an RNA-seq experiment derive predominantly from mRNA. One of the most commonly used commercial kits for rRNA depletion, the Ribo-Zero kit from Illumina, was recently discontinued. Here, we report the development a simple, cost-effective, and robust method for depleting rRNA that can be easily implemented by any lab or facility. We first developed an algorithm for designing biotinylated oligonucleotides that will hybridize tightly and specifically to the 23S, 16S, and 5S rRNAs from any species of interest. Precipitation of these oligonucleotides bound to rRNA by magnetic streptavidin beads then depletes rRNA from a complex, total RNA sample such that ~75-80% of reads in a typical RNA-seq experiment derive from mRNA. Importantly, we demonstrate a high correlation of RNA abundance or fold-change measurements in RNA-seq experiments between our method and the previously available Ribo-Zero kit. Complete details on the methodology are provided, including open-source software for designing oligonucleotides optimized for any bacterial species or metagenomic sample of interest.ImportanceThe ability to examine global patterns of gene expression in microbes through RNA-sequencing has fundamentally transformed microbiology. However, RNA-seq depends critically on the removal of ribosomal RNA from total RNA samples. Otherwise, rRNA would comprise upwards of 90% of the reads in a typical RNA-seq experiment, limiting the reads coming from messenger RNA or requiring high total read depth. A commonly used, kit for rRNA subtraction from Illumina was recently discontinued. Here, we report the development of a ‘do-it-yourself’ kit for rapid, cost-effective, and robust depletion of rRNA from total RNA. We present an algorithm for designing biotinylated oligonucleotides that will hybridize to the rRNAs from a target set of species. We then demonstrate that the designed oligos enable sufficient rRNA depletion to produce RNA-seq data with 75-80% of reads comming from mRNA. The methodology presented should enable RNA-seq studies on any species or metagenomic sample of interest.

scholarly journals Efficient depletion of ribosomal RNA for RNA sequencing in planarians

BMC Genomics ◽  
2019 ◽  
Vol 20 (1) ◽  
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

Abstract Background The astounding regenerative abilities of planarian flatworms prompt 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. Results In 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 from stem cells (termed neoblasts) of S. mediterranea and compared ribodepleted libraries with publicly available poly(A)-enriched ones. Overall, mRNA levels after ribodepletion were consistent 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. Conclusions The 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.

scholarly journals Overexpression of OsABCG48 Lowers Cadmium in Rice (Oryza sativa L.)

Agronomy ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 918
Author(s):  
Xingzhe Cai ◽  
Meng Wang ◽  
Yucong Jiang ◽  
Changhu Wang ◽  
David W. Ow
Keyword(s):  
Oryza Sativa L ◽  
Cost Effective ◽  
Health Issues ◽  
Rna Seq ◽  
Wild Type ◽  
Genetic Changes ◽  
Cd Accumulation ◽  
Lateral Root Development ◽  
Atp Binding Cassette Transporter ◽  
Cost Effective Approach

Cadmium pollution threatens food safety and security by causing health issues and reducing farmland availability. Engineering genetic changes in crop plants to lower Cd accumulation can be a cost-effective approach to address this problem. Previously, we reported that a rice line, 2B, which expresses a truncated version of OsO3L2 had reduced Cd accumulation throughout the plant, including in seed. However, downstream events caused by expression of this gene were not known. In this study, RNA-seq was used to identify differentially expressed genes between the wild type and 2B rice with or without Cd treatment, leading to the study of an ABC transporter gene, OsABCG48 (ATP-Binding Cassette transporter G family member 48). Heterologous expression of OsABCG48 conferred tolerance to Cd in Schizosaccharomyces pombe, Arabidopsis and rice. Moreover, overexpressing OsABCG48 in rice lowered root Cd accumulation that was associated with more extensive lateral root development. These data suggest that OsABCG48 might have applications for engineering low-Cd rice.

scholarly journals Gene Expression Imputation with Generative Adversarial Imputation Nets

2020 ◽  
Author(s):  
Ramon Viñas ◽  
Tiago Azevedo ◽  
Eric R. Gamazon ◽  
Pietro Liò
Keyword(s):  
Gene Expression ◽  
Large Scale ◽  
Biological Significance ◽  
Predictive Performance ◽  
Cost Effective ◽  
Rna Seq ◽  
Comprehensive Collection ◽  
Genomic Studies ◽  
Biological Discovery ◽  
Cancer Types

AbstractA question of fundamental biological significance is to what extent the expression of a subset of genes can be used to recover the full transcriptome, with important implications for biological discovery and clinical application. To address this challenge, we present GAIN-GTEx, a method for gene expression imputation based on Generative Adversarial Imputation Networks. In order to increase the applicability of our approach, we leverage data from GTEx v8, a reference resource that has generated a comprehensive collection of transcriptomes from a diverse set of human tissues. We compare our model to several standard and state-of-the-art imputation methods and show that GAIN-GTEx is significantly superior in terms of predictive performance and runtime. Furthermore, our results indicate strong generalisation on RNA-Seq data from 3 cancer types across varying levels of missingness. Our work can facilitate a cost-effective integration of large-scale RNA biorepositories into genomic studies of disease, with high applicability across diverse tissue types.

scholarly journals A practical guide to buildde-novoassemblies for single tissues of non-model organisms: the example of a Neotropical frog

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3702 ◽  
Author(s):  
Santiago Montero-Mendieta ◽  
Manfred Grabherr ◽  
Henrik Lantz ◽  
Ignacio De la Riva ◽  
Jennifer A. Leonard ◽  
...  
Keyword(s):  
Defense Mechanisms ◽  
De Novo ◽  
Transcriptome Assembly ◽  
Cost Effective ◽  
Model Organisms ◽  
Rna Seq ◽  
Assembly Pipeline ◽  
Wide Variability ◽  
History Of ◽  
Inexperienced User

Whole genome sequencing (WGS) is a very valuable resource to understand the evolutionary history of poorly known species. However, in organisms with large genomes, as most amphibians, WGS is still excessively challenging and transcriptome sequencing (RNA-seq) represents a cost-effective tool to explore genome-wide variability. Non-model organisms do not usually have a reference genome and the transcriptome must be assembledde-novo. We used RNA-seq to obtain the transcriptomic profile forOreobates cruralis, a poorly known South American direct-developing frog. In total, 550,871 transcripts were assembled, corresponding to 422,999 putative genes. Of those, we identified 23,500, 37,349, 38,120 and 45,885 genes present in the Pfam, EggNOG, KEGG and GO databases, respectively. Interestingly, our results suggested that genes related to immune system and defense mechanisms are abundant in the transcriptome ofO. cruralis. We also present a pipeline to assist with pre-processing, assembling, evaluating and functionally annotating ade-novotranscriptome from RNA-seq data of non-model organisms. Our pipeline guides the inexperienced user in an intuitive way through all the necessary steps to buildde-novotranscriptome assemblies using readily available software and is freely available at:https://github.com/biomendi/TRANSCRIPTOME-ASSEMBLY-PIPELINE/wiki.

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

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.

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

2011 ◽  
Vol 8 (11) ◽  
pp. iii-iv ◽  
Author(s):  
Vladimir Benes ◽  
Jonathon Blake ◽  
Ken Doyle
Keyword(s):  
Ribosomal Rna ◽  
Rna Seq

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

RNA ◽  
2020 ◽  
Vol 26 (11) ◽  
pp. 1731-1742 ◽  
Author(s):  
Mary Kay Thompson ◽  
Maria Kiourlappou ◽  
Ilan Davis
Keyword(s):  
Ribosomal Rna ◽  
Cost Effective ◽  
Rna Depletion

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

2020 ◽  
Vol 6 (3) ◽  
pp. 32 ◽  
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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