Targeted rRNA depletion enables efficient mRNA sequencing in diverse bacterial species and complex co-cultures.

Bacterial mRNA sequencing is inefficient due to the abundance of ribosomal RNA that is challenging to deplete. While commercial kits target rRNA from common bacterial species, they are frequently inefficient when applied to divergent species, including those from environmental isolates. Similarly, other methods typically employ large probe sets that tile the entire length of rRNAs; however, such approaches are infeasible when applied to many species. Therefore, we present EMBR-seq+, which requires fewer than 10 oligonucleotides per rRNA by combining rRNA blocking primers with RNase H-mediated depletion to achieve rRNA removal efficiencies of up to 99% in diverse bacterial species. Furthermore, in more complex microbial co-cultures between Fibrobacter succinogenes strain UWB7 and anaerobic fungi, EMBR-seq+ depleted both bacterial and fungal rRNA, with a fourfold improvement in bacterial rRNA depletion compared with a commercial kit, thereby demonstrating that the method can be applied to non-model microbial mixtures. Notably, for microbes with unknown rRNA sequences, EMBR-seq+ enables rapid iterations in probe design without requiring to start experiments from total RNA. Finally, efficient depletion of rRNA enabled systematic quantification of the reprogramming of the bacterial transcriptome when cultured in the presence of the anaerobic fungi Anaeromyces robustus or Caecomyces churrovis . We observed that F. succinogenes strain UWB7 downregulated several lignocellulose-degrading carbohydrate-active enzymes in the presence of anaerobic gut fungi, suggesting close interactions between two cellulolytic species that specialize in different aspects of biomass breakdown. Thus, EMBR-seq+ enables efficient, cost-effective, and rapid quantification of the transcriptome to gain insights into non-model microbial systems. IMPORTANCE Microbes present one of the most diverse sources of biochemistry in nature, and mRNA sequencing provides a comprehensive view of this biological activity by quantitatively measuring microbial transcriptomes. However, efficient mRNA capture for sequencing presents significant challenges in prokaryotes as mRNAs are not poly-adenylated and typically make up less than 5% of total RNA compared with rRNAs that exceed 80%. Recently developed methods for sequencing bacterial mRNA typically rely on depleting rRNA by tiling large probe sets against rRNAs; however, such approaches are expensive, time-consuming, and challenging to scale to varied bacterial species and complex microbial communities. Therefore, we developed EMBR-seq+, a method that requires fewer than 10 short oligonucleotides per rRNA to achieve up to 99% rRNA depletion in diverse bacterial species. Finally, EMBR-seq+ resulted in a deeper view of the transcriptome, enabling systematic quantification of how microbial interactions result in altering the transcriptional state of bacteria within co-cultures.