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

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, we describe a probe and ribonuclease based strategy for bacterial rRNA removal. We 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. We further showed that different probe sets can be used on closely related species. We 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.