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MDRIP-seq is a high-throughput method for quantitative R-loop landscape profiling

ABSTRACT: Current methods for R-loop profiling need to perform experiments for each sample individually, with consequent limitations in throughput. Here, based on the barcoding strategy, we develop mDRIP-seq, a high-throughput method showing equivalent performance as conventional methods, but with merits of 7-fold less cost and 6-fold less hand-on time per sample. We also show the simplicity and effectiveness of mDRIP-seq for relative and absolute quantitation of genomic R-loop fractions for multiple samples. Together, mDRIP-seq is a high-throughput and cost-efficient method for R-loop mapping and quantitative assessment that can be widely applied to large-scale dynamic profiles of these important structures for diverse organisms.

ORGANISM(S): Oryza sativa Escherichia coli Arabidopsis thaliana Mus musculus Saccharomyces cerevisiae Homo sapiens

PROVIDER: GSE219071 | GEO | 2024/06/11

REPOSITORIES: GEO

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Project description:Short-read DNA sequencing technologies provide new tools to answer biological questions. However, high cost and low throughput limit their widespread use, particularly in organisms with smaller genomes such as S. cerevisiae. Although ChIP-Seq in mammalian cell lines is replacing array-based ChIP-chip as the standard for transcription factor binding studies, ChIP-Seq in yeast is still underutilized compared to ChIP-chip. We developed a multiplex barcoding system that allows simultaneous sequencing and analysis of multiple samples using Illumina’s platform. We applied this method to analyze the chromosomal distributions of three yeast DNA binding proteins (Ste12, Cse4 and RNA PolII) and a reference sample (input DNA) in a single experiment and demonstrate its utility for rapid and accurate results at reduced costs. We developed a barcoding ChIP-Seq method for the concurrent analysis of transcription factor binding sites for yeast. Our multiplex strategy generated high quality data that was indistinguishable from data obtained with non-barcoded libraries. None of the barcoded adapters induced differences relative to a non-barcoded adapter when applied to the same DNA sample. We used this method to map the binding sites for Cse4, Ste12 and Pol II throughout the yeast genome and we found 148 binding targets for Cse4, 823 targets for Ste12 and 2508 targets for PolII. Cse4 was strongly bound to all yeast centromeres as expected and the remaining non-centromeric targets correspond to highly expressed genes in rich media, the latter constituting a novel finding. We designed a multiplex short-read DNA sequencing method to perform efficient ChIP-Seq in yeast and other small genome model organisms. This method produces accurate results with higher throughput and reduced cost. Given constant improvements in high-throughput sequencing technologies, increasing multiplexing will be possible to further decrease costs per sample and to accelerate the completion of large consortium projects such as modENCODE.

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MDRIP-seq is a high-throughput method for quantitative R-loop landscape profiling

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