Dataset Information

A reverse transcriptase-mediated ribosomal RNA depletion (RTR2D) strategy for the cost-effective construction of RNA sequencing libraries.

ABSTRACT: RNA sequencing (RNA-seq)-based whole transcriptome analysis (WTA) using ever-evolving next-generation sequencing technologies has become a primary tool for coding and/or noncoding transcriptome profiling. As WTA requires RNA-seq data for both coding and noncoding RNAs, one key step for obtaining high-quality RNA-seq data is to remove ribosomal RNAs, which can be accomplished by using various commercial kits. Nonetheless, an ideal rRNA removal method should be efficient, user-friendly and cost-effective so it can be adapted for homemade RNA-seq library construction. Here, we developed a novel reverse transcriptase-mediated ribosomal RNA depletion (RTR2D) method. We demonstrated that RTR2D was simple and efficient, and depleted human or mouse rRNAs with high specificity without affecting coding and noncoding transcripts. RNA-seq data analysis indicated that RTR2D yielded highly correlative transcriptome landscape with that of NEBNext rRNA Depletion Kit at both mRNA and lncRNA levels. In a proof-of-principle study, we found that RNA-seq dataset from RTR2D-depleted rRNA samples identified more differentially expressed mRNAs and lncRNAs regulated by Nutlin3A in human osteosarcoma cells than that from NEBNext rRNA Depletion samples, suggesting that RTR2D may have lower off-target depletion of non-rRNA transcripts. Collectively, our results have demonstrated that the RTR2D methodology should be a valuable tool for rRNA depletion.

SUBMITTER: Zeng Z

PROVIDER: S-EPMC7195496 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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A reverse transcriptase-mediated ribosomal RNA depletion (RTR2D) strategy for the cost-effective construction of RNA sequencing libraries.

Zeng Zongyue Z Huang Bo B Wang Xi X Fan Jiaming J Zhang Bo B Yang Lijuan L Feng Yixiao Y Wu Xiaoxing X Luo Huaxiu H Zhang Jing J Zhang Meng M He Fang F Mao Yukun Y Pakvasa Mikhail M Wagstaff William W Li Alexander J AJ Liu Bin B Ding Huimin H Zhang Yongtao Y Niu Changchun C Wu Meng M Zhao Xia X Wolf Jennifer Moriatis JM Lee Michael J MJ Huang Ailong A Luu Hue H HH Haydon Rex C RC He Tong-Chuan TC

Journal of advanced research 20200102

RNA sequencing (RNA-seq)-based whole transcriptome analysis (WTA) using ever-evolving next-generation sequencing technologies has become a primary tool for coding and/or noncoding transcriptome profiling. As WTA requires RNA-seq data for both coding and noncoding RNAs, one key step for obtaining high-quality RNA-seq data is to remove ribosomal RNAs, which can be accomplished by using various commercial kits. Nonetheless, an ideal rRNA removal method should be efficient, user-friendly and cost-ef ...[more]

PMID: 32373357

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Project description:The profiling of gene expression by RNA sequencing (RNA-seq) has enabled powerful studies of global transcriptional patterns in all organisms, including bacteria. Because the vast majority of RNA in bacteria is rRNA, it is standard practice to deplete the rRNA from a total RNA sample such that the reads in an RNA-seq experiment derive predominantly from mRNA. One of the most commonly used commercial kits for rRNA depletion, the Ribo-Zero kit from Illumina, was recently discontinued abruptly and for an extended period of time. Here, we report the development of a simple, cost-effective, and robust method for depleting rRNA that can be easily implemented by any lab or facility. We first developed an algorithm for designing biotinylated oligonucleotides that will hybridize tightly and specifically to the 23S, 16S, and 5S rRNAs from any species of interest. Precipitation of these oligonucleotides bound to rRNA by magnetic streptavidin-coated beads then depletes rRNA from a complex, total RNA sample such that ?75 to 80% of reads in a typical RNA-seq experiment derive from mRNA. Importantly, we demonstrate a high correlation of RNA abundance or fold change measurements in RNA-seq experiments between our method and the Ribo-Zero kit. Complete details on the methodology are provided, including open-source software for designing oligonucleotides optimized for any bacterial species or community of interest.IMPORTANCE The ability to examine global patterns of gene expression in microbes through RNA sequencing has fundamentally transformed microbiology. However, RNA-seq depends critically on the removal of rRNA from total RNA samples. Otherwise, rRNA would comprise upward of 90% of the reads in a typical RNA-seq experiment, limiting the reads coming from mRNA or requiring high total read depth. A commonly used kit for rRNA subtraction from Illumina was recently unavailable for an extended period of time, disrupting routine rRNA depletion. Here, we report the development of a "do-it-yourself" kit for rapid, cost-effective, and robust depletion of rRNA from total RNA. We present an algorithm for designing biotinylated oligonucleotides that will hybridize to the rRNAs from a target set of species. We then demonstrate that the designed oligonucleotides enable sufficient rRNA depletion to produce RNA-seq data with 75 to 80% of reads coming from mRNA. The methodology presented should enable RNA-seq studies on any species or metagenomic sample of interest.

Dataset Information

A reverse transcriptase-mediated ribosomal RNA depletion (RTR2D) strategy for the cost-effective construction of RNA sequencing libraries.

Publications

A reverse transcriptase-mediated ribosomal RNA depletion (RTR2D) strategy for the cost-effective construction of RNA sequencing libraries.

Similar Datasets

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