A MATQ-seq-Based Protocol for Single-Cell RNA-seq in Bacteria

Microbes exhibit an extraordinary capacity to adapt their physiology to different environments using phenotypic heterogeneity. However, the majority of gene regulation studies are conducted in bulk reflecting only averaged gene expression levels across millions of cells. Bacterial single-cell RNA-seq (scRNA-seq) can overcome this by enabling whole transcriptome and unbiased analysis of microbes at the single-cell level. Here, we describe a detailed workflow of single-cell RNA-seq based on the multiple annealing and dC-tailing-based quantitative single-cell RNA-seq (MATQ-seq) protocol. Following adjustments to the original eukaryotic protocol, the workflow was applied to two major human pathogens Salmonella enterica serovar Typhimurium (henceforth Salmonella) and Pseudomonas aeruginosa (henceforth Pseudomonas). The development of bacterial scRNA-seq protocols offers promising avenues to explore the molecular programs underlying phenotypic heterogeneity on the transcriptome level in different settings such as infection, persistence, ecology, and biofilms.