Efficient depletion of ribosomal RNA for RNA sequencing in planarians

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.

Download Full-text

Efficient depletion of ribosomal RNA for RNA sequencing in planarians

BMC Genomics ◽

10.1186/s12864-019-6292-y ◽

2019 ◽

Vol 20 (1) ◽

Cited By ~ 7

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.

Download Full-text

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

Download Full-text

Reduced miR-26b Expression in Megakaryocytes and Platelets Contributes to Elevated Level of Platelet Activation Status in Sepsis

International Journal of Molecular Sciences ◽

10.3390/ijms21030866 ◽

2020 ◽

Vol 21 (3) ◽

pp. 866 ◽

Cited By ~ 5

Author(s):

Bernadett Szilágyi ◽

Zsolt Fejes ◽

Szilárd Póliska ◽

Marianna Pócsi ◽

Zsolt Czimmerer ◽

...

Keyword(s):

Platelet Activation ◽

Mirna Expression ◽

Mrna Level ◽

Severe Infection ◽

Mrna Levels ◽

Rna Seq ◽

Calpain Inhibition ◽

The Impact ◽

Lps Treatment ◽

Activation Status

In sepsis, platelets may become activated via toll-like receptors (TLRs), causing microvascular thrombosis. Megakaryocytes (MKs) also express these receptors; thus, severe infection may modulate thrombopoiesis. To explore the relevance of altered miRNAs in platelet activation upon sepsis, we first investigated sepsis-induced miRNA expression in platelets of septic patients. The effect of abnormal Dicer level on miRNA expression was also evaluated. miRNAs were profiled in septic vs. normal platelets using TaqMan Open Array. We validated platelet miR-26b with its target SELP (P-selectin) mRNA levels and correlated them with clinical outcomes. The impact of sepsis on MK transcriptome was analyzed in MEG-01 cells after lipopolysaccharide (LPS) treatment by RNA-seq. Sepsis-reduced miR-26b was further studied using Dicer1 siRNA and calpain inhibition in MEG-01 cells. Out of 390 platelet miRNAs detected, there were 121 significantly decreased, and 61 upregulated in sepsis vs. controls. Septic platelets showed attenuated miR-26b, which were associated with disease severity and mortality. SELP mRNA level was elevated in sepsis, especially in platelets with increased mean platelet volume, causing higher P-selectin expression. Downregulation of Dicer1 generated lower miR-26b with higher SELP mRNA, while calpeptin restored miR-26b in MEG-01 cells. In conclusion, decreased miR-26b in MKs and platelets contributes to an increased level of platelet activation status in sepsis.

Download Full-text

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.

Download Full-text

Leucine Supplementation Decreases HDAC4 Expression and Nuclear Localization in Skeletal Muscle Fiber of Rats Submitted to Hindlimb Immobilization

Cells ◽

10.3390/cells9122582 ◽

2020 ◽

Vol 9 (12) ◽

pp. 2582

Author(s):

Paula K. N. Alves ◽

André Cruz ◽

William J. Silva ◽

Siegfried Labeit ◽

Anselmo S. Moriscot

Keyword(s):

Skeletal Muscle ◽

Target Genes ◽

Marked Decrease ◽

Mrna Levels ◽

Rna Seq ◽

Rat Skeletal Muscle ◽

Leucine Supplementation ◽

Male Wistar Rats ◽

Histone Deacetylase 4 ◽

The Impact

In this study we surveyed a rat skeletal muscle RNA-Seq for genes that are induced by hindlimb immobilization and, in turn, become attenuated by leucine supplementation. This approach, in search of leucine-atrophy protection mediating genes, identified histone deacetylase 4 (HDAC4) as highly responsive to both hindlimb immobilization and leucine supplementation. We then examined the impact of leucine on HDAC4 expression, tissue localization, and target genes. A total of 76 male Wistar rats (~280 g) were submitted to hindlimb immobilization and/or leucine supplementation for 3, 7 and 12 days. These animals were euthanized, and soleus muscle was removed for further analysis. RNA-Seq analysis of hindlimb immobilized rats indicated a sharp induction (log2 = 3.4) of HDAC4 expression which was attenuated by leucine supplementation (~50%). Real-time PCR and protein expression analysis by Western blot confirmed increased HDAC4 mRNA after 7 days of hindlimb immobilization and mitigation of induction by leucine supplementation. Regarding the HDAC4 localization, the proportion of positive nuclei was higher in the immobilized group and decreased after leucine supplementation. Also, we found a marked decrease of myogenin and MAFbx-atrogin-1 mRNA levels upon leucine supplementation, while CAMKII and DACH2 mRNA levels were increased by leucine supplementation. Our data suggest that HDAC4 inhibition might be involved in the anti-atrophic effects of leucine.

Download Full-text

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

10.1101/2020.01.06.896837 ◽

2020 ◽

Cited By ~ 4

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.

Download Full-text

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

10.21203/rs.3.rs-67621/v3 ◽

2021 ◽

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

Download Full-text

Testing the Genomic Shock Hypothesis Using Transposable Element Expression in Yeast Hybrids

Frontiers in Fungal Biology ◽

10.3389/ffunb.2021.729264 ◽

2021 ◽

Vol 2 ◽

Author(s):

Marika Drouin ◽

Mathieu Hénault ◽

Johan Hallin ◽

Christian R. Landry

Keyword(s):

Transposable Element ◽

Differential Expression Analysis ◽

Ribosome Profiling ◽

Control Mechanisms ◽

Translation Efficiency ◽

Mrna Levels ◽

Rna Seq ◽

Ty Elements ◽

Translational Activity ◽

The Impact

Transposable element (TE) insertions are a source of structural variation and can cause genetic instability and gene expression changes. A host can limit the spread of TEs with various repression mechanisms. Many examples of plant and animal interspecific hybrids show disrupted TE repression leading to TE propagation. Recent studies in yeast did not find any increase in transposition rate in hybrids. However, this does not rule out the possibility that the transcriptional or translational activity of TEs increases following hybridization because of a disruption of the host TE control mechanisms. Thus, whether total expression of a TE family is higher in hybrids than in their parental species remains to be examined. We leveraged publically available RNA-seq and ribosomal profiling data on yeast artificial hybrids of the Saccharomyces genus and performed differential expression analysis of their LTR retrotransposons (Ty elements). Our analyses of total mRNA levels show that Ty elements are generally not differentially expressed in hybrids, even when the hybrids are exposed to a low temperature stress condition. Overall, only 2/26 Ty families show significantly higher expression in the S. cerevisiae × S. uvarum hybrids while there are 3/26 showing significantly lower expression in the S. cerevisiae x S. paradoxus hybrids. Our analysis of ribosome profiling data of S. cerevisiae × S. paradoxus hybrids shows similar translation efficiency of Ty in both parents and hybrids, except for Ty1_cer showing higher translation efficiency. Overall, our results do not support the hypothesis that hybridization could act as a systematic trigger of TE expression in yeast and suggest that the impact of hybridization on TE activity is strain and TE specific.

Download Full-text

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

BMC Genomics ◽

10.1186/s12864-021-07381-z ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Benjamin P. Kellman ◽

Hratch M. 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. Graphical abstract

Download Full-text

Regulatory and sustainability initiatives lead to improved polyaminopolyamide epichlorohydrin (PAE) wet-strength resins and paper products

TAPPI Journal ◽

10.32964/tj17.09.519 ◽

2018 ◽

Vol 17 (09) ◽

pp. 519-532 ◽

Cited By ~ 1

Author(s):

Mark Crisp ◽

Richard Riehle

Keyword(s):

Health And Safety ◽

Cost Effective ◽

Worker Safety ◽

Regulatory Requirements ◽

Wet Strength ◽

The Past ◽

Consumer Safety ◽

Federal Institute ◽

Sustainability Initiatives ◽

The Impact

Polyaminopolyamide-epichlorohydrin (PAE) resins are the predominant commercial products used to manufacture wet-strengthened paper products for grades requiring wet-strength permanence. Since their development in the late 1950s, the first generation (G1) resins have proven to be one of the most cost-effective technologies available to provide wet strength to paper. Throughout the past three decades, regulatory directives and sustainability initiatives from various organizations have driven the development of cleaner and safer PAE resins and paper products. Early efforts in this area focused on improving worker safety and reducing the impact of PAE resins on the environment. These efforts led to the development of resins containing significantly reduced levels of 1,3-dichloro-2-propanol (1,3-DCP) and 3-monochloropropane-1,2-diol (3-MCPD), potentially carcinogenic byproducts formed during the manufacturing process of PAE resins. As the levels of these byproducts decreased, the environmental, health, and safety (EH&S) profile of PAE resins and paper products improved. Recent initiatives from major retailers are focusing on product ingredient transparency and quality, thus encouraging the development of safer product formulations while maintaining performance. PAE resin research over the past 20 years has been directed toward regulatory requirements to improve consumer safety and minimize exposure to potentially carcinogenic materials found in various paper products. One of the best known regulatory requirements is the recommendations of the German Federal Institute for Risk Assessment (BfR), which defines the levels of 1,3-DCP and 3-MCPD that can be extracted by water from various food contact grades of paper. These criteria led to the development of third generation (G3) products that contain very low levels of 1,3-DCP (typically <10 parts per million in the as-received/delivered resin). This paper outlines the PAE resin chemical contributors to adsorbable organic halogens and 3-MCPD in paper and provides recommendations for the use of each PAE resin product generation (G1, G1.5, G2, G2.5, and G3).

Download Full-text