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Multimodal RNA-seq using single-strand, double-strand, and circligase-based capture yields a refined and extended description of the C. elegans transcriptome

ABSTRACT: We have used a combination of three high-throughput RNA capture and sequencing methods to refine and augment the transcriptome map of a well studied genetic model, Caenorhabditis elegans. The three methods include a standard (non-directional) library preparation protocol relying on cDNA priming and foldback that has been used in several previous studies for transcriptome characterization in this species, and two directional protocols, one involving direct capture of single stranded RNA fragments and one involving circular-template PCR (circligase). We find that each RNA-seq approach shows specific limitations and biases, with the application of multiple methods providing a more complete map than was obtained from any single method. Of particular note in the analysis were substantial advantages of circligase-based and ssRNA-based capture for defining sequences and structures of the precise 5' ends (which were lost using the double strand cDNA capture method). Of the three methods, ssRNA capture was most effective in defining sequences to the polyA junction. Using datasets from a spectrum of C. elegans strains and stages and the UCSC Genome Browser, we provide a series of tools, which facilitate rapid visualization and assignment of gene structures.

ORGANISM(S): Caenorhabditis elegans

PROVIDER: GSE22410 | GEO | 2010/10/20

SECONDARY ACCESSION(S): PRJNA128465

REPOSITORIES: GEO

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Multimodal RNA-seq using single-strand, double-strand, and circligase-based capture yields a refined and extended description of the C. elegans transcriptome

Project description:We have used a combination of three high-throughput RNA capture and sequencing methods to refine and augment the transcriptome map of a well studied genetic model, Caenorhabditis elegans. The three methods include a standard (non-directional) library preparation protocol relying on cDNA priming and foldback that has been used in several previous studies for transcriptome characterization in this species, and two directional protocols, one involving direct capture of single stranded RNA fragments and one involving circular-template PCR (circligase). We find that each RNA-seq approach shows specific limitations and biases, with the application of multiple methods providing a more complete map than was obtained from any single method. Of particular note in the analysis were substantial advantages of circligase-based and ssRNA-based capture for defining sequences and structures of the precise 5' ends (which were lost using the double strand cDNA capture method). Of the three methods, ssRNA capture was most effective in defining sequences to the polyA junction. Using datasets from a spectrum of C. elegans strains and stages and the UCSC Genome Browser, we provide a series of tools, which facilitate rapid visualization and assignment of gene structures. single-strand-capture, double-strand-capture, and circligase-based RNA-seq

2010-10-20 | E-GEOD-22410 | biostudies-arrayexpress

Sequencing of First-strand cDNA Library Reveals Full-length Transcriptomes

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2015-01-01 | E-GEOD-63424 | biostudies-arrayexpress

Sequencing of First-strand cDNA Library Reveals Full-length Transcriptomes

2015-01-01 | GSE63424 | GEO

Global analysis of RNA secondary structure conservation between two metazoans

Project description:The secondary structure of RNA is necessary for its maturation, regulation, and processing. However, the global influence of RNA folding in eukaryotes is still unclear. Here, we identify evolutionarily conserved features of RNA secondary structure in metazoans by applying our high-throughput, sequencing-based, structure-mapping approach to Drosophila melanogaster and Caenorhabditis elegans. This analysis reveals key structural patterns across protein-coding transcripts that indicate RNA folding is influential to protein translation and microRNA-mediated targeting and/or regulation of mRNAs in animals. Additionally, we uncover a novel population of highly base-paired RNAs, many of which are likely functional, long, non-coding RNAs. Finally, we identify and characterize ~180 structural motifs of mRNAs that are under positive or negative selection in these metazoans, thereby revealing a large set of RNA structures that are likely functional. Overall, our findings highlight the significance of secondary structure within RNA molecules, and provide the first comprehensive evidence of widespread RNA secondary structure conservation in animals. Double-stranded (dsRNA) specific RNA sequencing (dsRNA-seq) and single-stranded (ssRNA) specific RNA sequencing (ssRNA-seq) in Drosophila DL1 cells and C. elegans mixed stage N2 worms. Each of the four samples (dsRNA/Dmel, ssRNA/Dmel, dsRNA/Cel, and ssRNA/Cel) was sequenced separately on both Illumina GA-IIx and Hiseq2000, giving a total of eight datasets. Two corresponding smRNA libraries (smRNA-seq) of the same DL1 cells and mixed stage N2 worms are also presented.

2012-01-26 | E-GEOD-29571 | biostudies-arrayexpress

Transposon-based construction of strand-specific RNA-seq libraries

Project description:We have developed two methods for efficiently consructing RNA-seq libraries using transposition. Each method constructs high quality RNA-seq libraries when compared to standard approaches. One of the methods (Directional Tn-RNA-seq) maintains strand-of-origin information and exhibits strand specificity comparable to current approaches. RNA-seq libraries were constructed from ECC-1, a human cell line, and Universal Human Reference RNA using transposon-based and standard RNA-seq library construciton methods.

2011-10-10 | E-GEOD-32307 | biostudies-arrayexpress

Transposon-based construction of strand-specific RNA-seq libraries

2011-10-10 | GSE32307 | GEO

unclassified ssRNA positive-strand viruses

Project description:unclassified ssRNA positive-strand viruses

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high efficiency and multiplexable Strand specificity RNA sequencing by Double Adapter Directional Capture

Project description:The quantification of transcriptome through RNA sequencing (RNA-seq) has widely been used to characterize all patterns of genes in a certain period. However, although many experimental protocols have recently been developed, RNA-seq is still a challenging technology in application. It is necessary to develop an easy and versatile RNA-seq method available in a wide range from single cell RNA to bulk RNA. Here we introduce a novel RNA-seq method, Double Adapter Directional Capture sequencing (DADCSeq), by directly adding adaptor at both termini of library respectively in the steps of reverse transcription and second strand synthesis, which can not only produce high quality strand specificity full-length RNA-seq library but also reduces cockamamie steps of library constructions with less time consuming. The DADCSeq was also optimized for prokaryote such as Mycobacterium smegmatis, a model bacterium for Mycobacterium tuberculosis, and can detect more genes compared to traditional methods. Taken together the DADCSeq offers a greatly simplified and cost-effective protocol with high repeatable strand specificity RNA-seq results for a diversity of RNA input materials from prokaryote to eucaryote RNA-seq libraries.

2023-07-23 | GSE237259 | GEO

Determination of RNA Secondary Structure By RNA-Sequencing of HEK293T Cells

Project description:RNA molecule fold into three dimensional structures in order to fill important roles in the cell such as translation and post-transcriptional regulation. These three dimensional structures, in turn, depend on the pattern of hydrogen bonds between ribonucleotides. We employ a technique based on high throughput RNA Sequencing (RNA-Seq) to uncover this patter on a transcriptome wide scale. We take advantage of two ribonucleases that have structure specific activity. RNAse S1 specifically digests single stranded RNA while RNAse V1 is specific to double stranded RNA. We aliquot each of our sample into to reactions. One is thoroughly treated with RNAse S1, digesting away single stranded RNA and leaving behind dsRNA. The other is treated with RNAse V1, which has the inverse effect. The surviving dsRNA and ssRNA molecules are then subject to high throughput sequencing. The resulting reads are used to generate a structure score, revelaing which regions of each transcript are involved in base pairing and which are unpaired.

2016-05-17 | E-GEOD-72681 | biostudies-arrayexpress

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Project description:We tried two methods which are the DNase I treated full-length double-strand cDNA sequencing and the poly(A) capture full-length double-strand cDNA sequencing to avoid the non-specific genomic DNA amplification.

2022-06-29 | GSE192928 | GEO

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