The State Key Laboratory Breeding Base of Basic Science of Stomatology & Key Laboratory of Oral Biomedicine Ministry of Education, School & Hospital of Stomatology, Medical Research Institute, Wuhan University, Wuhan, China; Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, China
Hebin Liao
The State Key Laboratory Breeding Base of Basic Science of Stomatology & Key Laboratory of Oral Biomedicine Ministry of Education, School & Hospital of Stomatology, Medical Research Institute, Wuhan University, Wuhan, China; Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, China; Translational Medicine Research Center, North Sichuan Medical College, Nanchong, China
Chenyi Wang
The State Key Laboratory Breeding Base of Basic Science of Stomatology & Key Laboratory of Oral Biomedicine Ministry of Education, School & Hospital of Stomatology, Medical Research Institute, Wuhan University, Wuhan, China; Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, China
Chun Huang
The State Key Laboratory Breeding Base of Basic Science of Stomatology & Key Laboratory of Oral Biomedicine Ministry of Education, School & Hospital of Stomatology, Medical Research Institute, Wuhan University, Wuhan, China; Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, China
Wei Zhang
The State Key Laboratory Breeding Base of Basic Science of Stomatology & Key Laboratory of Oral Biomedicine Ministry of Education, School & Hospital of Stomatology, Medical Research Institute, Wuhan University, Wuhan, China; Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, China
Chunming Guo
Center for Life Sciences, School of Life Sciences, Yunnan University, Kunming, China
The State Key Laboratory Breeding Base of Basic Science of Stomatology & Key Laboratory of Oral Biomedicine Ministry of Education, School & Hospital of Stomatology, Medical Research Institute, Wuhan University, Wuhan, China; Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, China; Department of Immunology, Hubei Province Key Laboratory of Allergy and Immunology, State Key Laboratory of Virology and Medical Research Institute, Wuhan University School of Basic Medical Sciences, Wuhan, China
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.
