scholarly journals Ancient RNA from Late Pleistocene permafrost and historical canids shows tissue-specific transcriptome survival

2019 ◽  
Author(s):  
Oliver Smith ◽  
Glenn Dunshea ◽  
Mikkel-Holger S. Sinding ◽  
Sergey Fedorov ◽  
Mietje Germonpre ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
High Throughput Sequencing ◽  
Rna Seq ◽  
Biologically Relevant ◽  
Archaeological Material ◽  
Tissue Identification ◽  
Mammalian Tissues ◽  
Sequencing Platforms ◽  
Genome Activity

AbstractWhile sequencing ancient DNA from archaeological material is now commonplace, very few attempts to sequence ancient transcriptomes have been made, even from typically stable deposition environments such as permafrost. This is presumably due to assumptions that RNA completely degrades relatively quickly, particularly when dealing with autolytic, nuclease-rich mammalian tissues. However, given the recent successes in sequencing ancient RNA (aRNA) from various sources including plants and animals, we suspect that these assumptions may be incorrect or exaggerated. To challenge the underlying dogma, we generated shotgun RNA data from sources that might normally be dismissed for such study. Here we present aRNA data generated from two historical wolf skins, and permafrost-preserved liver tissue of a 14,300-year-old Pleistocene canid. Not only is the latter the oldest RNA ever to be sequenced, but also shows evidence of biologically relevant tissue-specificity and close similarity to equivalent data derived from modern-day control tissue. Other hallmarks of RNA-seq data such as exon-exon junction presence and high endogenous ribosomal RNA content confirms our data’s authenticity. By performing independent technical replicates using two high-throughput sequencing platforms, we show not only that aRNA can survive for extended periods in mammalian tissues, but also that it has potential for tissue identification, and possibly further uses such as in vivo genome activity and adaptation, when sequenced using this technology.

scholarly journals Characterization of the rat developmental liver transcriptome

2013 ◽  
Vol 45 (8) ◽  
pp. 301-311 ◽  
Author(s):  
Richard H. Chapple ◽  
Polyana C. Tizioto ◽  
Kevin D. Wells ◽  
Scott A. Givan ◽  
JaeWoo Kim ◽  
...  
Keyword(s):  
Gene Expression ◽  
High Throughput Sequencing ◽  
Mitochondrial Gene ◽  
Probe Design ◽  
Rna Seq ◽  
Illumina Hiseq ◽  
Biologically Relevant ◽  
Gradual Process ◽  
Intergenic Regions ◽  
Sequencing Platforms

Gene regulation and transcriptome studies have been enabled by the development of RNA-Seq applications for high-throughput sequencing platforms. Next generation sequencing is remarkably efficient and avoids many issues inherent in hybridization-based microarray methodologies including the exon-specific dependence of probe design. Biologically relevant transcripts including messenger and regulatory RNAs may now be quantified and annotated regardless of whether they have previously been observed. We used RNA-Seq to investigate global patterns of gene expression in early developing rat liver. Liver samples from timed-pregnant Lewis rats were collected at six fetal and neonatal stages [embryonic day (E)14, E16, E18, E20, postnatal day (P)1, P7], transcripts were sequenced using an Illumina HiSeq 2000, and data analysis was performed with the Tuxedo software suite. Genes and isoforms differing in abundance were queried for enrichment within functionally related gene groups using the Functional Annotation Tool of the DAVID Bioinformatics Database. While hematopoietic gene expression is initiated by E14, hepatocyte maturation is a gradual process involving clusters of genes responsible for response to nutrients and enzymes responsible for glycolysis and fatty acid catabolism. Following birth, a large cluster of differentially abundant genes was enriched for mitochondrial gene expression and cholesterol synthesis indicating that by 1 wk of age, the liver is engaged in lipid sensing and bile production. Clustering results for differentially abundant genes and isoforms were similar with the greatest difference for the E14/E16 comparison. Finally, a bioinformatic approach was used to annotate 1,307 novel liver transcripts assembled from sequences that aligned to intergenic regions of the rat genome.

scholarly journals Unbiased, Genome-Wide In Vivo Mapping of Transcriptional Regulatory Elements Reveals Sex Differences in Chromatin Structure Associated with Sex-Specific Liver Gene Expression

2010 ◽  
Vol 30 (23) ◽  
pp. 5531-5544 ◽  
Author(s):  
Guoyu Ling ◽  
Aarathi Sugathan ◽  
Tali Mazor ◽  
Ernest Fraenkel ◽  
David J. Waxman
Keyword(s):  
High Throughput Sequencing ◽  
Strong Association ◽  
Regulatory Elements ◽  
Sequence Motifs ◽  
Transcriptional Regulatory Elements ◽  
Transcriptional Regulatory ◽  
Mammalian Tissues ◽  
Hypersensitive Sites ◽  
Regulatory Sites

ABSTRACT We have used a simple and efficient method to identify condition-specific transcriptional regulatory sites in vivo to help elucidate the molecular basis of sex-related differences in transcription, which are widespread in mammalian tissues and affect normal physiology, drug response, inflammation, and disease. To systematically uncover transcriptional regulators responsible for these differences, we used DNase hypersensitivity analysis coupled with high-throughput sequencing to produce condition-specific maps of regulatory sites in male and female mouse livers and in livers of male mice feminized by continuous infusion of growth hormone (GH). We identified 71,264 hypersensitive sites, with 1,284 showing robust sex-related differences. Continuous GH infusion suppressed the vast majority of male-specific sites and induced a subset of female-specific sites in male livers. We also identified broad genomic regions (up to ∼100 kb) showing sex-dependent hypersensitivity and similar patterns of GH responses. We found a strong association of sex-specific sites with sex-specific transcription; however, a majority of sex-specific sites were >100 kb from sex-specific genes. By analyzing sequence motifs within regulatory regions, we identified two known regulators of liver sexual dimorphism and several new candidates for further investigation. This approach can readily be applied to mapping condition-specific regulatory sites in mammalian tissues under a wide variety of physiological conditions.

scholarly journals EU-RNA-seq for in vivo labeling and high throughput sequencing of nascent transcripts

2021 ◽  
Vol 2 (3) ◽  
pp. 100651
Author(s):  
Katherine C. Palozola ◽  
Greg Donahue ◽  
Kenneth S. Zaret
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Rna Seq ◽  
In Vivo Labeling ◽  
Nascent Transcripts

scholarly journals Nuclease-mediated depletion biases in ribosome footprint profiling libraries

2020 ◽  
Author(s):  
Boris Zinshteyn ◽  
Jamie R Wangen ◽  
Boyang Hua ◽  
Rachel Green
Keyword(s):  
Ribosomal Rna ◽  
High Throughput Sequencing ◽  
Precise Determination ◽  
Ribosome Profiling ◽  
Rna Seq ◽  
Rna Sequences ◽  
Ribosomal Rna Sequences ◽  
Rna Fragments ◽  
Commercial Applications

AbstractRibosome footprint profiling is a high throughput sequencing based technique that provides detailed and global views of translation in living cells. An essential part of this technology is removal of unwanted, normally very abundant, ribosomal RNA sequences that dominate libraries and increase sequencing costs. The most effective commercial solution (Ribo-Zero) has been discontinued and a number of new, experimentally distinct commercial applications have emerged on the market. Here we evaluated several commercially available alternatives designed for RNA-seq of human samples and find them unsuitable for ribosome footprint profiling. We instead recommend the use of custom-designed biotinylated oligos, which were widely used in early ribosome profiling studies. Importantly, we warn that depletion solutions based on targeted nuclease cleavage significantly perturb the high-resolution information that can be derived from the data, and thus do not recommend their use for any applications that require precise determination of the ends of RNA fragments.

Screening and Identification of Key Genes for the Survival and Multiplication of Salmonella typhimurium in the Host

2019 ◽  
Author(s):  
Na Sun ◽  
Yanying Song ◽  
Cong Liu ◽  
Yu Dai ◽  
Peng Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Salmonella Typhimurium ◽  
Mammalian Cells ◽  
High Throughput Sequencing ◽  
Bacterial Load ◽  
Sequencing Analysis ◽  
Rna Seq ◽  
Virulence Attenuation ◽  
Key Genes

Abstract BackgroundSalmonella typhimurium is an important intracellular pathogen that poses a health threat to humans. The key to studying the pathogenesis of Salmonella is to clarify the mechanisms responsible for its survival and reproduction in macrophages. In this study, RNA was extracted from S. typhimurium reference strain (ATCC 14028) and S. typhimurium isolated from the spleen of infected mice for RNA high-throughput sequencing analysis, based on the BGISEQ-500 platform.ResultsA total of 1,340 significant differentially expressed genes (DEGs) were screened through quantitative gene analysis and various analyses based on gene expression. Of these, 16 genes were randomly selected for fluorescence quantitative PCR verification. Two pairs of primers, 16S and pyridoxol 5ʹ-phosphate synthase (pdxJ), were used as internal references. The coincidence rate was determined to be 93.75%, which was consistent with the RNA-seq data, proving the reliability of the RNA-seq data. Functional annotation revealed DEGs associated with regulation, metabolism, transport and binding, pathogenesis, and motility. Through data mining and literature retrieval, 26 of the 58 upregulated DEGs (FPKM >10) were not reported to be related to the adaptation to intracellular survival, and were classified as candidate key genes (CKGs) for survival and proliferation in vivo. Among the CKGs, five were biotin synthetic bio family proteins. BioF is one of the first enzymes in the protein synthesis pathway. To evaluate the potential role of bioF in survival and proliferation, bioF mutants of Salmonella were constructed, and the virulence/attenuation was evaluated in vivo. Through the infection of BALB/c mice, bioF deficiency was found to significantly reduce the bacterial load and the fatality rate of mice. ConclusionsOur results indicated that the bioF gene plays an important role in the survival and proliferation of S. typhimurium in vivo. These data contribute to our understanding of the mechanisms used by Salmonella to regulate virulence gene expression whilst replicating inside mammalian cells.

scholarly journals Conditional Hfq Association with Small Noncoding RNAs in Pseudomonas aeruginosa Revealed through Comparative UV Cross-Linking Immunoprecipitation Followed by High-Throughput Sequencing

mSystems ◽  
2019 ◽  
Vol 4 (6) ◽  
Author(s):  
Kotaro Chihara ◽  
Thorsten Bischler ◽  
Lars Barquist ◽  
Vivian A. Monzon ◽  
Naohiro Noda ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
High Throughput Sequencing ◽  
Noncoding Rnas ◽  
Cross Linking ◽  
Rna Seq ◽  
Content Type ◽  
Total Rna ◽  
Small Noncoding Rnas ◽  
Rna Chaperones

ABSTRACT Bacterial small noncoding RNAs (sRNAs) play posttranscriptional regulatory roles in cellular responses to changing environmental cues and in adaptation to harsh conditions. Generally, the RNA-binding protein Hfq helps sRNAs associate with target mRNAs to modulate their translation and to modify global RNA pools depending on physiological state. Here, a combination of in vivo UV cross-linking immunoprecipitation followed by high-throughput sequencing (CLIP-seq) and total RNA-seq showed that Hfq interacts with different regions of the Pseudomonas aeruginosa transcriptome under planktonic versus biofilm conditions. In the present approach, P. aeruginosa Hfq preferentially interacted with repeats of the AAN triplet motif at mRNA 5′ untranslated regions (UTRs) and sRNAs and U-rich sequences at rho-independent terminators. Further transcriptome analysis suggested that the association of sRNAs with Hfq is primarily a function of their expression levels, strongly supporting the notion that the pool of Hfq-associated RNAs is equilibrated by RNA concentration-driven cycling on and off Hfq. Overall, our combinatorial CLIP-seq and total RNA-seq approach highlights conditional sRNA associations with Hfq as a novel aspect of posttranscriptional regulation in P. aeruginosa. IMPORTANCE The Gram-negative bacterium P. aeruginosa is ubiquitously distributed in diverse environments and can cause severe biofilm-related infections in at-risk individuals. Although the presence of a large number of putative sRNAs and widely conserved RNA chaperones in this bacterium implies the importance of posttranscriptional regulatory networks for environmental fluctuations, limited information is available regarding the global role of RNA chaperones such as Hfq in the P. aeruginosa transcriptome, especially under different environmental conditions. Here, we characterize Hfq-dependent differences in gene expression and biological processes in two physiological states: the planktonic and biofilm forms. A combinatorial comparative CLIP-seq and total RNA-seq approach uncovered condition-dependent association of RNAs with Hfq in vivo and expands the potential direct regulatory targets of Hfq in the P. aeruginosa transcriptome.

scholarly journals Organism-specific depletion of highly abundant RNA species from bacterial total RNA

2020 ◽  
Vol 2 (10) ◽  
Author(s):  
Florian Engelhardt ◽  
Jürgen Tomasch ◽  
Susanne Häussler
Keyword(s):  
Ribosomal Rna ◽  
Messenger Rna ◽  
Type Species ◽  
High Throughput Sequencing ◽  
Rnase H ◽  
Rna Seq ◽  
Content Type ◽  
Standard Tool ◽  
Cost Efficient ◽  
Host Infection

High-throughput sequencing has become a standard tool for transcriptome analysis. The depletion of overrepresented RNA species from sequencing libraries plays a key role in establishing potent and cost-efficient RNA-seq routines. Commercially available kits are known to obtain good results for the reduction of ribosomal RNA (rRNA). However, we found that the transfer-messenger RNA (tmRNA) was frequently highly abundant in rRNA-depleted samples of Pseudomonas aeruginosa , consuming up to 25 % of the obtained reads. The tmRNA fraction was particularly high in samples taken from stationary cultures. This suggests that overrepresentation of this RNA species reduces the mRNA fraction when cells are grown under challenging conditions. Here, we present an RNase-H-based depletion protocol that targets the tmRNA in addition to ribosomal RNAs. We were able to increase the mRNA fraction to 93–99% and therefore outperform not only the commercially Ribo-off kit (Vazyme) operating by the same principle but also the formerly widely used Ribo-Zero kit (Illumina). Maximizing the read share of scientifically interesting RNA species enhances the discriminatory potential of next-generation RNA-seq experiments and, therefore, can contribute to a better understanding of the transcriptomic landscape of bacterial pathogens and their used mechanisms in host infection.

scholarly journals Transcriptome Profile Alterations with Carbon Nanotubes, Quantum Dots, and Silver Nanoparticles: A Review

Genes ◽  
2021 ◽  
Vol 12 (6) ◽  
pp. 794
Author(s):  
Cullen Horstmann ◽  
Victoria Davenport ◽  
Min Zhang ◽  
Alyse Peters ◽  
Kyoungtae Kim
Keyword(s):  
Carbon Nanotubes ◽  
Quantum Dots ◽  
High Throughput Sequencing ◽  
The Body ◽  
Rna Seq ◽  
Mechanisms Of Toxicity ◽  
Promising Tool ◽  
Engineered Nanomaterial ◽  
Next Generation Sequencing Ngs ◽  
Transcriptomic Changes

Next-generation sequencing (NGS) technology has revolutionized sequence-based research. In recent years, high-throughput sequencing has become the method of choice in studying the toxicity of chemical agents through observing and measuring changes in transcript levels. Engineered nanomaterial (ENM)-toxicity has become a major field of research and has adopted microarray and newer RNA-Seq methods. Recently, nanotechnology has become a promising tool in the diagnosis and treatment of several diseases in humans. However, due to their high stability, they are likely capable of remaining in the body and environment for long periods of time. Their mechanisms of toxicity and long-lasting effects on our health is still poorly understood. This review explores the effects of three ENMs including carbon nanotubes (CNTs), quantum dots (QDs), and Ag nanoparticles (AgNPs) by cross examining publications on transcriptomic changes induced by these nanomaterials.

scholarly journals BRD4 PROTAC degrader ARV-825 inhibits T-cell acute lymphoblastic leukemia by targeting 'Undruggable' Myc-pathway genes

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Shuiyan Wu ◽  
You Jiang ◽  
Yi Hong ◽  
Xinran Chu ◽  
Zimu Zhang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Acute Lymphoblastic Leukemia ◽  
Caspase 3 ◽  
Lymphoblastic Leukemia ◽  
Rna Seq ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Bet Protein

Abstract Background T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease with a high risk of induction failure and poor outcomes, with relapse due to drug resistance. Recent studies show that bromodomains and extra-terminal (BET) protein inhibitors are promising anti-cancer agents. ARV-825, comprising a BET inhibitor conjugated with cereblon ligand, was recently developed to attenuate the growth of multiple tumors in vitro and in vivo. However, the functional and molecular mechanisms of ARV-825 in T-ALL remain unclear. This study aimed to investigate the therapeutic efficacy and potential mechanism of ARV-825 in T-ALL. Methods Expression of the BRD4 were determined in pediatric T-ALL samples and differential gene expression after ARV-825 treatment was explored by RNA-seq and quantitative reverse transcription-polymerase chain reaction. T-ALL cell viability was measured by CCK8 assay after ARV-825 administration. Cell cycle was analyzed by propidium iodide (PI) staining and apoptosis was assessed by Annexin V/PI staining. BRD4, BRD3 and BRD2 proteins were detected by western blot in cells treated with ARV-825. The effect of ARV-825 on T-ALL cells was analyzed in vivo. The functional and molecular pathways involved in ARV-825 treatment of T-ALL were verified by western blot and chromatin immunoprecipitation (ChIP). Results BRD4 expression was higher in pediatric T-ALL samples compared with T-cells from healthy donors. High BRD4 expression indicated a poor outcome. ARV-825 suppressed cell proliferation in vitro by arresting the cell cycle and inducing apoptosis, with elevated poly-ADP ribose polymerase and cleaved caspase 3. BRD4, BRD3, and BRD2 were degraded in line with reduced cereblon expression in T-ALL cells. ARV-825 had a lower IC50 in T-ALL cells compared with JQ1, dBET1 and OTX015. ARV-825 perturbed the H3K27Ac-Myc pathway and reduced c-Myc protein levels in T-ALL cells according to RNA-seq and ChIP. In the T-ALL xenograft model, ARV-825 significantly reduced tumor growth and led to the dysregulation of Ki67 and cleaved caspase 3. Moreover, ARV-825 inhibited cell proliferation by depleting BET and c-Myc proteins in vitro and in vivo. Conclusions BRD4 indicates a poor prognosis in T-ALL. The BRD4 degrader ARV-825 can effectively suppress the proliferation and promote apoptosis of T-ALL cells via BET protein depletion and c-Myc inhibition, thus providing a new strategy for the treatment of T-ALL.

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