Department of Chemical Engineering, University of California Santa Barbara, Santa Barbara, California, USA.
Biological Engineering Program, University of California Santa Barbara, Santa Barbara, California, USA.
mSystems. 2023 Dec 21;8(6):e0028123. doi: 10.1128/msystems.00281-23. Epub 2023 Oct 19.
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.
微生物是自然界中生物化学多样性最丰富的来源之一，而 mRNA 测序通过定量测量微生物转录组，提供了对这种生物活性的全面观察。然而，在原核生物中，高效的 mRNA 捕获测序存在显著挑战，因为与超过 80%的 rRNA 相比，mRNA 不被聚腺苷酸化，通常只占总 RNA 的不到 5%。最近开发的用于测序细菌 mRNA 的方法通常依赖于通过针对 rRNA 的大片段探针耗尽 rRNA；然而，这种方法昂贵、耗时，并且难以扩展到不同的细菌物种和复杂的微生物群落。因此，我们开发了 EMBR-seq+，这是一种方法，每个 rRNA 需要不到 10 个短寡核苷酸即可实现高达 99%的 rRNA 耗尽，适用于多种细菌物种。最后，EMBR-seq+ 提供了转录组的更深入视图，能够系统地定量研究微生物相互作用如何导致共培养物中细菌转录状态的改变。
