Recent advances in single-cell RNA sequencing of bacteria: Techniques, challenges, and applications

Single-cell RNA sequencing (scRNA-seq) has revolutionized our understanding of cellular heterogeneity in complex biological systems. While this technology has been widely applied to eukaryotic cells, its adaptation to bacterial systems has been challenging due to the unique characteristics of bacterial transcripts. This review surveys the recent developments in bacterial scRNA-seq techniques, highlighting the technical challenges, methodological innovations, and emerging applications in microbiology. We discuss the key differences between eukaryotic and bacterial RNA-seq approaches, focusing on the strategies to overcome limitations such as the lack of poly-A tails in bacterial mRNAs and the low RNA content in individual bacterial cells. The review covers various bacterial scRNA-seq methods, including plate-based, split-pool barcoding, and droplet-based techniques, comparing their strengths and limitations in terms of sensitivity, throughput, and applicability to different bacterial species. Furthermore, we explore the biological insights gained from these techniques, such as identifying rare cell states, characterization of antibiotic responses, and analysis of bacterial communities. Finally, we discuss future perspectives and potential applications of bacterial scRNA-seq in understanding microbial physiology, host-pathogen interactions, and complex microbial ecosystems. This comprehensive overview aims to provide researchers with a clear understanding of the current state and future directions of single-cell transcriptomics in bacteria.