Abstract Clonal bacterial populations rely on transcriptional variation to differentiate into specialized cell states that increase the community’s fitness. Such heterogeneous gene expression is implicated in many fundamental microbial processes including sporulation, cell communication, detoxification, substrate utilization, competence, biofilm formation, motility, pathogenicity, and antibiotic resistance 1 . To identify these specialized cell states and determine the processes by which they develop, we need to study isogenic bacterial populations at the single cell level 2,3 . Here, we develop a method that uses DNA probes and leverages an existing commercial microfluidic platform (10X Chromium) to conduct bacterial single cell RNA sequencing. We sequenced the transcriptome of over 15,000 individual bacterial cells, detecting on average 365 transcripts mapping to 265 genes per cell in B. subtilis and 329 transcripts mapping to 149 genes per cell in E. coli . Our findings correctly identify known cell states and uncover previously unreported cell states. Interestingly, we find that some metabolic pathways segregate into distinct subpopulations across different bacteria and growth conditions, suggesting that some cellular processes may be more prone to differentiation than others. Our high throughput, highly resolved single cell transcriptomic platform can be broadly used for understanding heterogeneity in microbial populations.

Droplet-based single cell RNA sequencing of bacteria identifies known and previously unseen cellular states