Profiling the transcriptome with RNA SPOTs.
Ontology highlight
ABSTRACT:
SUBMITTER: Eng CL
PROVIDER: S-EPMC5819366 | biostudies-literature | 2017 Dec
REPOSITORIES: biostudies-literature
Publications
Profiling the transcriptome with RNA SPOTs.
Eng Chee-Huat Linus CL Shah Sheel S Thomassie Julian J Cai Long L
Nature methods 20171113 12
Single-molecule FISH (smFISH) has been the gold standard for quantifying individual transcript abundances. Here, we scale up multiplexed smFISH to the transcriptome level and profile 10,212 different mRNAs from mouse fibroblast and embryonic stem cells. This method, called RNA sequential probing of targets (SPOTs), provides an accurate, flexible, and low-cost alternative to sequencing for profiling transcriptomes. ...[more]
Similar Datasets
Project description:Accurate profiling of minute quantities of RNA in a global manner can enable key advances in many scientific and clinical disciplines. Here, we present low-quantity RNA sequencing (LQ-RNAseq), a high-throughput sequencing-based technique allowing whole transcriptome surveys from subnanogram RNA quantities in an amplification/ligation-free manner. LQ-RNAseq involves first-strand cDNA synthesis from RNA templates, followed by 3' polyA tailing of the single-stranded cDNA products and direct single molecule sequencing. We applied LQ-RNAseq to profile S. cerevisiae polyA+ transcripts, demonstrate the reproducibility of the approach across different sample preparations and independent instrument runs, and establish the absolute quantitative power of this method through comparisons with other reported transcript profiling techniques and through utilization of RNA spike-in experiments. We demonstrate the practical application of this approach to define the transcriptional landscape of mouse embryonic and induced pluripotent stem cells, observing transcriptional differences, including over 100 genes exhibiting differential expression between these otherwise very similar stem cell populations. This amplification-independent technology, which utilizes small quantities of nucleic acid and provides quantitative measurements of cellular transcripts, enables global gene expression measurements from minute amounts of materials and offers broad utility in both basic research and translational biology for characterization of rare cells.
| S-EPMC2847755 | biostudies-literature
Project description:RNAs can fold into compact three-dimensional structures, and most RNAs undergo protein interactions in the cell. These compact and occluded environments can block the ability of structure-probing agents to provide useful data about the folding and modification of the underlying RNA. The development of probes that can analyze structure in crowded settings, and differentiate the proximity of interactions, can shed new light on RNA biology. To this end, here we employ 2'-OH-reactive probes that are small enough to access folded RNA structure underlying many close molecular contacts within cells, providing considerably broader coverage for intracellular RNA structural analysis. We compare reverse transcriptase stops in RNA-Seq data from probes of small and standard size to assess RNA-protein proximity and evaluate solvent-exposed tunnels adjacent to RNA. The data are analyzed first with structurally characterized complexes (human 18S and 28S RNA), and then applied transcriptome-wide to polyadenylated transcripts in HEK293 cells. In our transcriptome profile, the smallest probe acetylimidazole (AcIm) yields 80% greater structural coverage than larger conventional reagent NAIN3, providing enhanced structural information in hundreds of transcripts. We further show that acetyl probes provide superior signals for identifying m6A modification sites in transcripts, and provide information regarding methylation sites that are inaccessible to a larger standard probe. RNA infrastructure profiling (RISP) enables enhanced analysis of transcriptome structure, modification, and interactions in living cells, especially in spatially crowded settings.
| S-EPMC10592667 | biostudies-literature
Project description:PurposeTranscriptome is the entire repertoire of transcripts present in a cell at any particular time. We undertook a next-generation whole transcriptome sequencing approach to gain insight into the transcriptional landscape of the developing mouse lens.MethodsWe ascertained mouse lenses at six developmental time points including two embryonic (E15 and E18) and four postnatal stages (P0, P3, P6, and P9). The ocular tissue at each time point was maintained as two distinct pools serving as biological replicates for each developmental stage. The mRNA and small RNA libraries were paired-end sequenced on Illumina HiSeq 2000 and subsequently analyzed using bioinformatics tools.ResultsMapping of mRNA and small RNA libraries generated 187.56 and 154.22 million paired-end reads, respectively. We detected a total of 14,465 genes in the mouse ocular lens at the above-mentioned six developmental stages. Of these, 46 genes exhibited a 40-fold differential (higher or lower) expression at one the five developmental stages (E18, P0, P3, P6, and P9) compared with their expression level at E15. Likewise, small RNA profiling identified 379 microRNAs (miRNAs) expressed in mouse lens at six developmental time points. Of these, 49 miRNAs manifested an 8-fold differential (higher or lower) expression at one the five developmental stages, as mentioned above compared with their expression level at E15.ConclusionsWe report a comprehensive profile of developing murine lens transcriptome including both mRNA and miRNA through next-generation RNA sequencing. A complete repository of the lens transcriptome of six developmental time points will be monumental in elucidating processes essential for the development of the ocular lens and maintenance of its transparency.
| S-EPMC4525677 | biostudies-literature
Project description:RNA sequencing has greatly facilitated gene expression studies but is weak in studying temporal RNA dynamics; this issue can be addressed by analyzing nascent RNAs. A famous method for nascent RNA analysis is metabolic labeling with noncanonic nucleoside followed by affinity purification, however, purification processes can always introduce biases into data analysis. Here, a chemical method for nascent RNA sequencing that avoids affinity purification based on acrylonitrile-mediated uridine-to-cytidine (U-to-C) conversion (AMUC-seq) via 4-thiouridine (s4U) cyanoethylation is presented. This method converts s4U base-pairing with guanine through the nucleophilic addition of s4U to acrylonitrile. The high reaction efficiency permits AMUC-seq directly and efficiently to recover nascent RNA information from total RNAs. AMUC-seq is validated by being used to detect mRNA half-lives and investigating the direct gene targets of a G-quadruplex stabilizer, which can be regarded as potential anticancer drug, in human cells. Thousands of direct gene targets of this drug are verified (these genes are significantly enriched in cancer such as SRC and HRAS). AMUC-seq also confirms G-quadruplex stabilization that impacts RNA polyadenylation. These results show AMUC-seq is qualified for the study of temporal RNA dynamics, and it can be a promising strategy to study the therapeutic mechanism of transcription-modulating drugs.
| S-EPMC7175251 | biostudies-literature
Project description:Memory programming of cytotoxic T cells (CTLs) by inflammatory cytokines can be regulated by mammalian target of rapamycin (mTOR). We have shown that inhibition of mTOR during CTL activation leads to the enhancement of memory, but the molecular mechanisms remain largely unknown. Using high-throughput RNA-Seq, we identified genes and functions in mouse CTLs affected by mTOR inhibition through rapamycin. Of the 43,221 identified transcripts, 184 transcripts were differentially expressed after rapamycin treatment, corresponding to 128 annotated genes. Of these genes, 114 were downregulated and only 14 were upregulated. Most importantly, 50 of them are directly related to cell death and survival. In addition, several genes such as CD62L are related to migration. Furthermore, we predicted downregulation of transcriptional regulators based on the total differentially expressed genes, as well as the subset of apoptosis-related genes. Quantitative PCR confirmed the differential expressions detected in RNA-Seq. We conclude that the regulatory function of rapamycin may work through inhibition of multiple genes related to apoptosis and migration, which enhance CTL survival into memory.
| S-EPMC4198470 | biostudies-literature
Project description:BACKGROUND:Suaeda glauca, a succulent halophyte of the Chenopodiaceae family, is widely distributed in coastal areas of China. Suaeda glauca is highly resistant to salt and alkali stresses. In the present study, the salt-responsive transcriptome of Suaeda glauca was analyzed to identify genes involved in salt tolerance and study halophilic mechanisms in this halophyte. RESULTS:Illumina HiSeq 2500 was used to sequence cDNA libraries from salt-treated and control samples with three replicates each treatment. De novo assembly of the six transcriptomes identified 75,445 unigenes. A total of 23,901 (31.68%) unigenes were annotated. Compared with transcriptomes from the three salt-treated and three salt-free samples, 231 differentially expressed genes (DEGs) were detected (including 130 up-regulated genes and 101 down-regulated genes), and 195 unigenes were functionally annotated. Based on the Gene Ontology (GO), Clusters of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) classifications of the DEGs, more attention should be paid to transcripts associated with signal transduction, transporters, the cell wall and growth, defense metabolism and transcription factors involved in salt tolerance. CONCLUSIONS:This report provides a genome-wide transcriptional analysis of a halophyte, Suaeda glauca, under salt stress. Further studies of the genetic basis of salt tolerance in halophytes are warranted.
| S-EPMC4773115 | biostudies-literature
Project description:BackgroundRNA-seq and microarray are the two popular methods employed for genome-wide transcriptome profiling. Current comparison studies have shown that transcriptome quantified by these two methods correlated well. However, none of them have addressed if they complement each other, considering the strengths and the limitations inherent with them. The pivotal requirement to address this question is the knowledge of a well known data set. In this regard, HrpX regulome from pathogenic bacteria serves as an ideal choice as the target genes of HrpX transcription factor are well studied due to their central role in pathogenicity.ResultsWe compared the performance of RNA-seq and microarray in their ability to detect known HrpX target genes by profiling the transcriptome from the wild-type and the hrpX mutant strains of γ-Proteobacterium Xanthomonas citri subsp. citri. Our comparative analysis indicated that gene expression levels quantified by RNA-seq and microarray well-correlated both at absolute as well as relative levels (Spearman correlation-coefficient, rs > 0.76). Further, the expression levels quantified by RNA-seq and microarray for the significantly differentially expressed genes (DEGs) also well-correlated with qRT-PCR based quantification (rs = 0.58 to 0.94). Finally, in addition to the 55 newly identified DEGs, 72% of the already known HrpX target genes were detected by both RNA-seq and microarray, while, the remaining 28% could only be detected by either one of the methods.ConclusionsThis study has significantly advanced our understanding of the regulome of the critical transcriptional factor HrpX. RNA-seq and microarray together provide a more comprehensive picture of HrpX regulome by uniquely identifying new DEGs. Our study demonstrated that RNA-seq and microarray complement each other in transcriptome profiling.
| S-EPMC3534599 | biostudies-literature
Project description:Most RNAs within polarized cells such as neurons are sorted subcellularly in a coordinated manner. Despite advances in the development of methods for profiling polyadenylated RNAs from small amounts of input RNA, techniques for profiling coding and non-coding RNAs simultaneously are not well established. Here, we optimized a transcriptome profiling method based on double-random priming and applied it to serially diluted total RNA down to 10 pg. Read counts of expressed genes were robustly correlated between replicates, indicating that the method is both reproducible and scalable. Our transcriptome profiling method detected both coding and long non-coding RNAs sized >300 bases. Compared to total RNAseq using a conventional approach our protocol detected 70% more genes due to reduced capture of ribosomal RNAs. We used our method to analyze the RNA composition of compartmentalized motoneurons. The somatodendritic compartment was enriched for transcripts with post-synaptic functions as well as for certain nuclear non-coding RNAs such as 7SK. In axons, transcripts related to translation were enriched including the cytoplasmic non-coding RNA 7SL. Our profiling method can be applied to a wide range of investigations including perturbations of subcellular transcriptomes in neurodegenerative diseases and investigations of microdissected tissue samples such as anatomically defined fiber tracts.
| S-EPMC4770199 | biostudies-literature
Project description:Giardia intestinalis is a common cause of diarrheal disease and it consists of eight genetically distinct genotypes or assemblages (A-H). Only assemblages A and B infect humans and are suggested to represent two different Giardia species. Correlations exist between assemblage type and host-specificity and to some extent symptoms. Phenotypical differences have been documented between assemblages and genome sequences are available for A, B and E. We have characterized and compared the polyadenylated transcriptomes of assemblages A, B and E. Four genetically different isolates were studied (WB (AI), AS175 (AII), P15 (E) and GS (B)) using paired-end, strand-specific RNA-seq. Most of the genome was transcribed in trophozoites grown in vitro, but at vastly different levels. RNA-seq confirmed many of the present annotations and refined the current genome annotation. Gene expression divergence was found to recapitulate the known phylogeny, and uncovered lineage-specific differences in expression. Polyadenylation sites were mapped for over 70% of the genes and revealed many examples of conserved and unexpectedly long 3' UTRs. 28 open reading frames were found in a non-transcribed gene cluster on chromosome 5 of the WB isolate. Analysis of allele-specific expression revealed a correlation between allele-dosage and allele expression in the GS isolate. Previously reported cis-splicing events were confirmed and global mapping of cis-splicing identified only one novel intron. These observations can possibly explain differences in host-preference and symptoms, and it will be the basis for further studies of Giardia pathogenesis and biology.
| S-EPMC3610916 | biostudies-literature
Project description:The molecular mechanisms underlying neuronal death are poorly understood. One of the most widely used models to study neuronal death are cultured cerebellar granule neurons (CGNs) which undergo apoptosis when switched from a medium containing depolarizing levels of potassium (HK) to a medium with low non-depolarizing levels of potassium (LK). Previously, other labs have used DNA microarray analysis to characterize gene expression changes in LK-treated CGNs. However, microarray analysis is only capable of measuring the status of known transcripts, and expression of low-abundance mRNAs is often not detected by the hybridization-based approach. We have used RNA-sequencing to conduct a more detailed and comprehensive analysis of gene expression changes in CGNs induced to die by LK treatment. RNA-seq investigates the status of both known transcripts as well as exploring new ones and is substantially more sensitive than the microarray approach. We have found that the expression of 4334 genes is significantly altered in LK-treated CGNs with 2199 being up-regulated while 2135 are down-regulated. Genes functioning in cell death and survival regulation, cell growth and proliferation and molecular transport were most affected by LK treatment. Further, a large number of genes involved in nervous system development and function were also deregulated. Analysis of signaling pathways that were affected in LK-induced death included but were not limited to mitochondrial dysfunction and oxidative phosphorylation, consistent with a number of studies showing perturbations of these pathways in neurodegenerative disorders. Thus, our study identifies a large number of new genes that are affected during the process of neuronal death. While a majority of these changes may reflect consequences of the induction of neuronal death, many of the genes that we have identified are likely to be critical and potentially novel mediators of neuronal death, including death associated with neurodegenerative disease.
| S-EPMC4640449 | biostudies-literature