3′-end poly(A)+ sequencing is an efficient and economical method for global measurement of mRNA levels and alternative poly(A) site usage. A common method involves oligo(dT)₁₉V reverse-transcription (RT)-based library preparation and high-throughput sequencing with a custom primer ending in (dT)₁₉. While the majority of library products have the first sequenced nucleotide reflect the bona fide poly(A) site (pA), a substantial fraction of sequencing reads arise from various mis-priming events. These can result in incorrect pA site calls anywhere from several nucleotides downstream to several kilobases upstream from the bona fide pA site. While these mis-priming events can be mitigated by increasing annealing stringency (e.g. increasing temperature from 37 °C to 42 °C), they still persist at an appreciable level (∼10%) and computational methods must be used to prevent artifactual calls. Here we present a bioinformatics workflow for precise mapping of poly(A)+ 3′ ends and handling of artifacts due to oligo(dT) mis-priming and sample polymorphisms. We test pA site calling with three different read mapping programs (STAR, BWA, and BBMap), and show that the way in which each handles terminal mismatches and soft clipping has a substantial impact on identifying correct pA sites, with BWA requiring the least post-processing to correct artifacts. We demonstrate the use of this pipeline for mapping pA sites in the model eukaryote S. cerevisiae, and further apply this technology to non-polyadenylated transcripts by employing in vitro polyadenylation prior to library prep (IVP-seq). As proof of principle, we show that a fraction of tRNAs harbor CCU 3′ tails instead of the canonical CCA tail, and globally identify 3′ ends of splicing intermediates arising from inefficiently spliced transcripts.

Features QuantSeq 3’ mRNA-Seq Library Prep Kit REV for Illumina

The eukaryotic translation initiation factor eIF4E is nuclear and cytoplasmic where it plays roles in export and translation of specific transcripts, respectively. When we were studying its mRNA export activity, we unexpectedly discovered that eIF4E drives the protein expression of elements of the 3′-end core cleavage complex involved in cleavage and polyadenylation (CPA), including CPSF3, the enzyme responsible for cleavage, as well as its co-factors CPSF1, CPSF2, CPSF4, Symplekin, WDR33, and FIP1L1. Using multiple strategies, we demonstrate that eIF4E stimulates 3′-end cleavage of selected RNAs. eIF4E physically interacts with CPSF3, CPSF1, and uncleaved target RNA, suggesting it acts directly and indirectly on the pathway. Through these effects, eIF4E can generate better substrates for its mRNA export and translation activities. Thus, we identified an unanticipated function for eIF4E in 3′-end processing of specific target RNAs, and this function could potentially affect the expression of a broad range of oncoproteins.

Features QuantSeq 3’ mRNA-Seq Library Prep Kit REV for Illumina

Cellular RNA levels are determined by the rates of RNA transcription from the gene template and subsequent RNA stability. Knowledge about both transcription and RNA decay is, therefore, necessary to interpret RNA levels and gene expression, especially during cellular processes where these parameters change. Numerous experimental strategies have been developed to measure transcription and RNA decay rates. However, to our knowledge, none of those techniques can simultaneously interrogate transcription and RNA decay. The presented protocol allows this and provides a simple approach to simultaneously estimate total RNA levels, transcription and decay rates from the same RNA sample. It is based on brief metabolic labeling of RNA and subsequent concurrent sequencing of polyA⁺ and polyA^– RNA 3′ ends. The protocol was developed in S. cerevisiae and should be broadly applicable.

Features QuantSeq 3’ mRNA-Seq Library Prep Kit REV for Illumina

Extensive tracts of the mammalian genome that lack protein-coding function are still transcribed into long noncoding RNA. While these lncRNAs are generally short lived, length restricted, and non-polyadenylated, how their expression is distinguished from protein-coding genes remains enigmatic. Surprisingly, depletion of the ubiquitous Pol-II-associated transcription elongation factor SPT6 promotes a redistribution of H3K36me3 histone marks from active protein coding to lncRNA genes, which correlates with increased lncRNA transcription. SPT6 knockdown also impairs the recruitment of the Integrator complex to chromatin, which results in a transcriptional termination defect for lncRNA genes. This leads to the formation of extended, polyadenylated lncRNAs that are both chromatin restricted and form increased levels of RNA:DNA hybrid (R-loops) that are associated with DNA damage. Additionally, these deregulated lncRNAs overlap with DNA replication origins leading to localized DNA replication stress and a cellular senescence phenotype. Overall, our results underline the importance of restricting lncRNA expression.

Features QuantSeq 3’ mRNA-Seq Library Prep Kit REV for Illumina

Niclosamide is an antihelminthic drug used worldwide for the treatment of tapeworm infections. Recent drug repurposing screens have revealed that niclosamide exhibits diverse mechanisms of action and, as a result, demonstrates promise for a number of applications, including the treatment of cancer, bacterial infections, and Zika virus. As new applications of niclosamide will require non-oral delivery routes that may lead to exposure in utero, the objective of this study was to investigate the mechanism of niclosamide toxicity during early stages of embryonic development. Using zebrafish as a model, we found that niclosamide induced a concentration-dependent delay in epiboly progression during late-blastula and early-gastrula, an effect that was dependent on exposure during the maternal-to-zygotic transition – a period characterized by degradation of maternally-derived transcripts, zygotic genome activation, and initiation of cell motility. Moreover, we found that niclosamide did not affect embryonic oxygen consumption, suggesting that oxidative phosphorylation – a well-established target for niclosamide within intestinal parasites – may not play a role in niclosamide-induced epiboly delay. However, mRNA-sequencing revealed that niclosamide exposure during blastula and early-gastrula significantly impacted the timing of zygotic genome activation as well as the abundance of cytoskeleton- and cell cycle regulation-specific transcripts. In addition, we found that niclosamide inhibited tubulin polymerization in vitro, suggesting that niclosamide-induced delays in epiboly progression may, in part, be driven by disruption of microtubule formation and cell motility within the developing embryo.

Features QuantSeq 3’ mRNA-Seq Library Prep Kit REV for Illumina

Expansin increases cell wall extensibility to allow cell wall loosening and cell expansion even in the absence of hydrolytic activity. Previous studies showed that excessive overexpression of expansin gene resulted in defective growth (Goh et al., 2014; Rochange et al., 2001) [1,2] and altered cell wall chemical composition (Zenoni et al., 2011) [3]. However, the molecular mechanism on how the overexpression of non-enzymatic cell wall protein expansin can result in widespread effects on plant cell wall and organ growth remains unclear. We acquired transcriptomic data on previously reported transgenic Arabidopsis line (Goh et al., 2014) [1] to investigate the effects of overexpressing a heterologus cucumber expansin gene (CsEXPA1) on the global gene expression pattern during early and late phases of etiolated hypocotyl growth.

Features QuantSeq 3’ mRNA-Seq Library Prep Kit REV for Illumina

Alzheimer’s disease (AD) involves changes in both lipid and RNA metabolism, but it remained unknown if these differences associate with AD’s cognition and/or post-mortem neuropathology indices. Here, we report RNA-sequencing evidence of inter-related associations between lipid processing, cognition level, and AD neuropathology. In two unrelated cohorts, we identified pathway-enriched facilitation of lipid processing and alternative splicing genes, including the neuronal-enriched NOVA1 and hnRNPA1. Specifically, this association emerged in temporal lobe tissue samples from donors where postmortem evidence demonstrated AD neuropathology, but who presented normal cognition proximate to death. The observed changes further associated with modified ATP synthesis and mitochondrial transcripts, indicating metabolic relevance; accordingly, mass-spectrometry-derived lipidomic profiles distinguished between individuals with and without cognitive impairment prior to death. In spite of the limited group sizes, tissues from persons with both cognitive impairment and AD pathology showed elevation in several drug-targeted genes of other brain, vascular and autoimmune disorders, accompanied by pathology-related increases in distinct lipid processing transcripts, and in the RNA metabolism genes hnRNPH2, TARDBP, CLP1 and EWSR1. To further detect 3′-polyadenylation variants, we employed multiple cDNA primer pairs. This identified variants that showed limited differences in scope and length between the tested cohorts, yet enabled superior clustering of demented and non-demented AD brains versus controls compared to total mRNA expression values. Our findings indicate inter-related cognition-associated differences in AD’s lipid processing, alternative splicing and 3′-polyadenylation, calling for pursuing the underlying psychological and therapeutics implications.

Features QuantSeq 3’ mRNA-Seq Library Prep Kit REV for Illumina and SPLIT RNA Extraction Kit

Papaya is considered to be one of the most nutritional fruits. It is rich in vitamins, carotenoids, flavonoids and other phytonutrient which function as antioxidant in our body [1]. Previous studies revealed that the suppression of a negative regulator gene in photomorphogenesis, De-etiolated 1 (DET1) can improve the phytonutrient in tomato and canola without affecting the fruit quality [2] ; [3]. This report contains the experimental data on high-throughput 3′ mRNA sequencing of transformed papaya callus upon DET1 gene suppression.

Features QuantSeq 3’ mRNA-Seq Library Prep Kit REV for Illumina

Expansin is a cell wall loosening protein without hydrolytic activity, which allows cell expansion by influencing cell wall extensibility. Previous studies showed that the suppression of expansin genes (EXPA1, EXPA3, EXPA5 and EXPA10) resulted in defective organ growth and altered cell wall chemical composition [1,2]. However, the molecular mechanism on how the suppression of non-enzymatic expansin expression can result in widespread effects on plant cell wall and organ growth is still unclear. In this study, we performed transcriptomic analysis on the hypocotyls of previously reported transgenic Arabidopsis line [1] to investigate the effects of expansin gene suppression on the global gene expression pattern, particularly on the cell wall related genes.

Features QuantSeq 3’ mRNA-Seq Library Prep Kit REV for Illumina

Many RNA-binding proteins (RBPs) regulate both alternative exons and poly(A) site selection. To understand their regulatory principles, we developed expressRNA, a web platform encompassing computational tools for integration of iCLIP and RNA motif analyses with RNA-seq and 3′ mRNA sequencing. This reveals at nucleotide resolution the “RNA maps” describing how the RNA binding positions of RBPs relate to their regulatory functions. We use this approach to examine how TDP-43, an RBP involved in several neurodegenerative diseases, binds around its regulated poly(A) sites. Binding close to the poly(A) site generally represses, whereas binding further downstream enhances use of the site, which is similar to TDP-43 binding around regulated exons. Our RNAmotifs2 software also identifies sequence motifs that cluster together with the binding motifs of TDP-43. We conclude that TDP-43 directly regulates diverse types of pre-mRNA processing according to common position-dependent principles.

Features QuantSeq 3’ mRNA-Seq Library Prep Kit REV for Illumina

In model organisms, classical genetic screening via random mutagenesis provides key insights into the molecular bases of genetic interactions, helping to define synthetic lethality, synthetic viability and drug-resistance mechanisms. The limited genetic tractability of diploid mammalian cells, however, precludes this approach. Here, we demonstrate the feasibility of classical genetic screening in mammalian systems by using haploid cells, chemical mutagenesis and next-generation sequencing, providing a new tool to explore mammalian genetic interactions.

Features QuantSeq 3’ mRNA-Seq Library Prep Kit REV for Illumina

Reverse genetic screens have driven gene annotation and target discovery in model organisms. However, many disease‐relevant genotypes and phenotypes cannot be studied in lower organisms. It is therefore essential to overcome technical hurdles associated with large‐scale reverse genetics in human cells. Here, we establish a reverse genetic approach based on highly robust and sensitive multiplexed RNA sequencing of mutant human cells. We conduct 10 parallel screens using a collection of engineered haploid isogenic cell lines with knockouts covering tyrosine kinases and identify known and unexpected effects on signaling pathways. Our study provides proof of concept for a scalable approach to link genotype to phenotype in human cells, which has broad applications. In particular, it clears the way for systematic phenotyping of still poorly characterized human genes and for systematic study of uncharacterized genomic features associated with human disease.

Features QuantSeq 3’ mRNA-Seq Library Prep Kit REV for Illumina

Genome-wide transcriptome profiling is a powerful tool to study global gene expression patterns in plant development. We report the first transcriptome profile analysis of papaya embryogenic callus to improve our understanding on genes associated with somatic embryogenesis. By using 3′ mRNA-sequencing, we generated 6,190,687 processed reads and 47.0% were aligned to papaya genome reference, in which 21,170 (75.4%) of 27,082 annotated genes were found to be expressed but only 41% was expressed at functionally high levels. The top 10% of genes with high transcript abundance were significantly enriched in biological processes related to cell proliferation, stress response, and metabolism. Genes functioning in somatic embryogenesis such as SERK and LEA, hormone-related genes, stress-related genes, and genes involved in secondary metabolite biosynthesis pathways were highly expressed. Transcription factors such as NAC, WRKY, MYB, WUSCHEL, Agamous-like MADS-box protein and bHLH important in somatic embryos of other plants species were found to be expressed in papaya embryogenic callus. Abundant expression of enolase and ADH is consistent with proteome study of papaya somatic embryo. Our study highlights that some genes related to secondary metabolite biosynthesis, especially phenylpropanoid biosynthesis, were highly expressed in papaya embryogenic callus, which might have implication for cell factory applications. The discovery of all genes expressed in papaya embryogenic callus provides an important information into early biological processes during the induction of embryogenesis and useful for future research in other plant species.

Features QuantSeq 3’ mRNA-Seq Library Prep Kit REV for Illumina

We present here the hypothesis that alternative poly-adenylation (APA) is dysregulated in the brains of individuals affected by Autism Spectrum Disorder (ASD), due to disruptions in the calcium signaling networks. APA, the process of selecting different poly-adenylation sites on the same gene, yielding transcripts with different-length 3′ untranslated regions (UTRs), has been documented in different tissues, stages of development and pathologic conditions. Differential use of poly-adenylation sites has been shown to regulate the function, stability, localization and translation efficiency of target RNAs. However, the role of APA remains rather unexplored in neurodevelopmental conditions. In the human brain, where transcripts have the longest 3′ UTRs and are thus likely to be under more complex post-transcriptional regulation, erratic APA could be particularly detrimental. In the context of ASD, a condition that affects individuals in markedly different ways and whose symptoms exhibit a spectrum of severity, APA dysregulation could be amplified or dampened depending on the individual and the extent of the effect on specific genes would likely vary with genetic and environmental factors. If this hypothesis is correct, dysregulated APA events might be responsible for certain aspects of the phenotypes associated with ASD. Evidence supporting our hypothesis is derived from standard RNA-seq transcriptomic data but we suggest that future experiments should focus on techniques that probe the actual poly-adenylation site (3′ sequencing). To address issues arising from the use of post-mortem tissue and low numbers of heterogeneous samples affected by confounding factors (such as the age, gender and health of the individuals), carefully controlled in vitro systems will be required to model the effect of calcium signaling dysregulation in the ASD brain.

Features QuantSeq 3’ mRNA-Seq Library Prep Kit REV for Illumina