In contrast to mammalian cells, bacterial cells lack mRNA polyadenylated tails, presenting a hurdle in isolating mRNA amidst the prevalent rRNA during single-cell RNA-seq. This study introduces a novel method, ribosomal RNA-derived cDNA depletion (RiboD), seamlessly integrated into the PETRI-seq technique, yielding RiboD-PETRI. This innovative approach offers a cost-effective, equipment-free, and high-throughput solution for bacterial single-cell RNA sequencing (scRNA-seq). By efficiently eliminating rRNA reads and substantially enhancing mRNA detection rates (up to 92%), our method enables precise exploration of bacterial population heterogeneity. Applying RiboD-PETRI to investigate biofilm heterogeneity, distinctive subpopulations marked by unique genes within biofilms were successfully identified. Notably, PdeI, a marker for the cell-surface attachment subpopulation, was observed to elevate cyclic diguanylate (c-di-GMP) levels, promoting persister cell formation. Thus, we address a persistent challenge in bacterial single-cell RNA-seq regarding rRNA abundance, exemplifying the utility of this method in exploring biofilm heterogeneity. Our method effectively tackles a long-standing issue in bacterial scRNA-seq: the overwhelming abundance of rRNA. This advancement significantly enhances our ability to investigate the intricate heterogeneity within biofilms at unprecedented resolution.
An improved bacterial single-cell RNA-seq reveals biofilm heterogeneity.
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作者:Yan Xiaodan, Liao Hebin, Wang Chenyi, Huang Chun, Zhang Wei, Guo Chunming, Pu Yingying
期刊: | Elife | 影响因子: | 6.400 |
时间: | 2024 | 起止号: | 2024 Dec 17; 13:RP97543 |
doi: | 10.7554/eLife.97543 |
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