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A cost-effective RNA sequencing protocol for large-scale gene expression studies.

ABSTRACT: RNA sequencing has increasingly become an indispensable tool for biological research. While sequencing costs have fallen dramatically in recent years, the current cost of RNA sequencing, nonetheless, remains a barrier to even more widespread adoption. Here, we present a simple RNA sequencing protocol with substantially reduced costs. This protocol uses as little as 10?ng of total RNA, allows multiplex sequencing of up to 96 samples per lane, and is strand specific. Extensive validation using human embryonic stem cells showed high consistency between technical replicates at various multiplexing levels.

SUBMITTER: Hou Z

PROVIDER: S-EPMC4381617 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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A cost-effective RNA sequencing protocol for large-scale gene expression studies.

Hou Zhonggang Z Jiang Peng P Swanson Scott A SA Elwell Angela L AL Nguyen Bao Kim S BK Bolin Jennifer M JM Stewart Ron R Thomson James A JA

Scientific reports 20150401

RNA sequencing has increasingly become an indispensable tool for biological research. While sequencing costs have fallen dramatically in recent years, the current cost of RNA sequencing, nonetheless, remains a barrier to even more widespread adoption. Here, we present a simple RNA sequencing protocol with substantially reduced costs. This protocol uses as little as 10 ng of total RNA, allows multiplex sequencing of up to 96 samples per lane, and is strand specific. Extensive validation using hum ...[more]

PMID: 25831155

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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 cost-effective RNA sequencing protocol for large-scale gene expression studies.

Publications

A cost-effective RNA sequencing protocol for large-scale gene expression studies.

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