Microbes in nature remain one of the largest untapped resources for novel bio-based products, including fuels, antibiotics, and waste treatment solutions. Delineating the transcriptomes of these species with RNA-seq would provide powerful datasets that could be used to characterize the cellular and biochemical functions within these species. However, a major challenge in bacterial RNA-seq is the removal of highly abundant ribosomal RNA (rRNA) molecules prior to sequencing. If left undepleted, rRNA can consume up to 90% of the sequencing space, resulting in highly inefficient sequencing of messenger RNA (mRNA), that encodes proteins. We previously developed a method, EMBR-seq, to deplete 5S, 16S, and 23S rRNA in E. coli, and showed that it effectively captures mRNA from as little as 0.02 ng of total RNA. Here, we report an improved version, EMBR-seq+, which depletes rRNA to 3% of sequenced reads with a combination of selective polyadenylation on mRNA and targeted RNase H activity on rRNA, improving the rRNA depletion efficiency two-fold over our previous report. We also apply EMBR-seq+ to other species, observing that rRNA was depleted to 18% of sequenced reads in C. acetobutylicum and 29% in G. metallireducens, demonstrating efficient RNA-seq of non-model prokaryotic species. Compared to alternate rRNA depletion methods that employ a large array of oligonucleotides, EMBR-seq+ employs a few oligonucleotides per rRNA subunit at strategic locations, thereby making this new technology significantly simpler. This method also integrates seamlessly into single-cell applications because the reverse transcription primers introduce cell-specific barcodes and the T7 promoter, enabling linear amplification of the template by in-vitro transcription and downstream single-cell demultiplexing. Thus, EMBR-seq+ provides an efficient approach to perform RNA-seq quantification of diverse species.

Efficient bacterial mRNA sequencing through targeted rRNA depletion

ACS Spring 2022

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