Scalable RNase H based rRNA depletion for bacterial transcriptomics

Bacterial RNA-seq is a powerful methodology to quantitatively understand the physiology and functionality of diverse microbial species. Depletion of ribosomal RNA (rRNA) is required to enable cost-effective sequencing of resulting libraries at appropriate coverage. However, current methodology for efficient rRNA depletion of diverse and non-model bacterial species is limited. Here we develop a new probe and RNase H based strategy for bacterial rRNA depletion. We validate the technique on 3 novel gut microbiota isolates representing three distinct phyla, show compatibility with chemically synthesized oligo or amplicon-based ssDNA probes, and demonstrate that probe sets can be successfully utilized on closely related species. A detailed protocol and online tool to generate probe libraries and predict performance on related species is available, and lays the groundwork for large-scale bacterial transcriptomics studies.