Project description:Paired-end deep-sequencing was used to determine in parallel the status and position of sister-chromatid contact (SC2) reporters and/or site-specific recombinase (SSR) activity reporters inserted at random positions in a library of cells. The SC2 reporters are short recombination cassettes for a topology-independent site-specific recombinase (Cre or Xer). The SSR-activity reporters are long recombination cassettes. The SC2 reporter cassettes are composed of two directly-repeated recombination sites separated by a DNA segment too short to permit their excision by intramolecular recombination. The SSR-activity cassettes are composed of two directly-repeated recombination sites separated by a DNA segment long enough to permit their excision by intramolecular recombination. The assays start with the engineering of a cell line with a conditional expression allele for Cre or Xer and the creation of a library of cells harbouring a cognate SC2 or SSR-activity reporter at different genomic positions. Production of the recombinase was induced for different lengths of time during cell growth and/or at specific stages of the cell cycle. The position of the SC2 reporter harboured by each cell and the recombination status of the recombination cassette it contains are then determined by high-throughput paired-end sequencing.
Project description:Studies have shown that Respiratory Burst Oxidase Homolog B (RBOHB) are involved in stress response in rice plants. Primers were developed for amplification via Polymerase Chain Reaction (PCR) of a region that contained a simple sequence repeat (SSR) in RBOHB. PCR was performed on 6 different varieties of Oryza sativa. PCR product was sequenced on an ABI 3730 capillary sequence machine. Sequence data was aligned to observe differences in SSR length between each rice variety.
Project description:Studies have shown that Rice Salt Sensitive 1 (RSS1) is involved in stress response in rice plants. Primers were developed for amplification via Polymerase Chain Reaction (PCR) of a region that contained a simple sequence repeat (SSR) in RSS1. PCR was performed on 6 different varieties of Oryza sativa. PCR product was sequenced on an ABI 3730 capillary sequence machine. Sequence data was aligned to observe differences in SSR length between each rice variety.
Project description:The advent of Next Generation Sequencing has allowed transcriptomes to be profiled with unprecedented accuracy, but the steep costs associated with full-length library sequencing have posed a limit on the accessibility and scalability of the technology. To address these limitations, we developed 3’Pool-seq, a simple, cost-effective, and scalable RNA-seq method that focuses sequencing to the 3’ end of mRNA transcripts.
Project description:The advent of Next Generation Sequencing has allowed transcriptomes to be profiled with unprecedented accuracy, but the steep costs associated with full-length library sequencing have posed a limit on the accessibility and scalability of the technology. To address these limitations, we developed 3’Pool-seq, a simple, cost-effective, and scalable RNA-seq method that focuses sequencing to the 3’ end of mRNA transcripts.
Project description:The advent of Next Generation Sequencing has allowed transcriptomes to be profiled with unprecedented accuracy, but the steep costs associated with full-length library sequencing have posed a limit on the accessibility and scalability of the technology. To address these limitations, we developed 3’Pool-seq, a simple, cost-effective, and scalable RNA-seq method that focuses sequencing to the 3’ end of mRNA transcripts.
Project description:The advent of Next Generation Sequencing has allowed transcriptomes to be profiled with unprecedented accuracy, but the steep costs associated with full-length library sequencing have posed a limit on the accessibility and scalability of the technology. To address these limitations, we developed 3’Pool-seq, a simple, cost-effective, and scalable RNA-seq method that focuses sequencing to the 3’ end of mRNA transcripts.
Project description:RNA-Seq provides an effective way to annotate and measure the transcriptome, but the combination of cost, analysis and end-mapping challenges has limited its adoption for high-throughput quantitation and annotation. Here, we present an integrated experimental and computational suite for transcriptome annotation and quantitation based on the sequencing of mRNA ends. It consists of: a novel, simple, one-step, strategy 5' RNA-Seq; an optimized library construction method for strand-specific 3' RNA-seq that reduces costs and time; and a complete computational analysis toolkit. We demonstrate the power and versatility of our approach to study the transcriptome of LPS stimulation in mouse dendritic cells, promoter structure of the TCRb locus in mouse T cells, and gene expression in circadian cycles in Drosophila. Our platform provides a comprehensive solution for high-throughput, cost-effective transcriptome quantification and end annotation. A study of 5' and 3' end RNA sequencing methods
Project description:RNA-Seq provides an effective way to annotate and measure the transcriptome, but the combination of cost, analysis and end-mapping challenges has limited its adoption for high-throughput quantitation and annotation. Here, we present an integrated experimental and computational suite for transcriptome annotation and quantitation based on the sequencing of mRNA ends. It consists of: a novel, simple, one-step, strategy 5' RNA-Seq; an optimized library construction method for strand-specific 3' RNA-seq that reduces costs and time; and a complete computational analysis toolkit. We demonstrate the power and versatility of our approach to study the transcriptome of LPS stimulation in mouse dendritic cells, promoter structure of the TCRb locus in mouse T cells, and gene expression in circadian cycles in Drosophila. Our platform provides a comprehensive solution for high-throughput, cost-effective transcriptome quantification and end annotation.
