Scalable and cost-effective ribonuclease-based rRNA depletion for transcriptomics

Area of Science:

• Microbiology
• Genomics
• Molecular Biology

Background:

• Bacterial RNA sequencing (RNA-seq) is vital for understanding microbial physiology and function.
• High ribosomal RNA (rRNa) abundance in bacterial samples complicates transcriptomic analysis.
• Existing methods for rRNA depletion are often ineffective for diverse, non-model bacterial species.

Purpose of the Study:

• To develop and validate a novel, cost-effective method for bacterial ribosomal RNA (rRNA) removal.
• To enable comprehensive transcriptomic profiling of diverse bacterial species, including non-model organisms.

Main Methods:

• A probe and ribonuclease-based strategy was designed for targeted rRNA depletion.
• Chemically synthesized oligonucleotides or amplicon-based single-stranded DNA probes were utilized.
• The method was validated on novel gut microbiota isolates from three distinct bacterial phyla.

Main Results:

• The developed method demonstrated effective removal of rRNA from diverse bacterial species.
• The technique proved adaptable, with different probe sets applicable to closely related species.
• A comprehensive protocol, extensive probe sets for over 5000 microbes, and an online probe design tool were provided.

Conclusions:

• This probe-based rRNA depletion strategy significantly enhances the feasibility of cost-effective bacterial RNA-seq.
• The approach facilitates large-scale transcriptomic studies of microbial communities.
• The provided resources lower the barrier for microbial transcriptomics research.