Bacterial RNA sequencing (RNA-seq) is a powerful approach for quantitatively delineating the global transcriptional profiles of microbes in order to gain deeper understanding of their physiology and function. Cost-effective bacterial RNA-seq requires efficient physical removal of ribosomal RNA (rRNA), which otherwise dominates transcriptomic reads. However, current methods to effectively deplete rRNA of diverse non-model bacterial species are lacking.
Here, Columbia University researchers describe a probe and ribonuclease based strategy for bacterial rRNA removal. They implemented the method using either chemically synthesized oligonucleotides or amplicon-based single-stranded DNA probes and validated the technique on three novel gut microbiota isolates from three distinct phyla. The researchers further showed that different probe sets can be used on closely related species. They provide a detailed methods protocol, probe sets for >5000 common microbes from RefSeq, and an online tool to generate custom probe libraries. This approach lays the groundwork for large-scale and cost-effective bacterial transcriptomics studies.
Workflow for bacterial RNase H based rRNA depletion
(A) Probes used for depletion can be either designed and chemically synthesized from known rRNA sequences (oligo-based) or generated by PCR from genomic DNA with 5′-phosphorylated forward primers and subsequent lambda exonuclease digestion (amplicon-based). (B) Probes are then hybridized to total RNA and the rRNA bound by the ssDNA probes is degraded by RNase H. At last, all remaining probes are degraded by DNase I or removed by SPRI beads-based size selection, resulting in enriched mRNAs.