Abstract
Bacterial populations are highly adaptive, enabling them to respond to and surviving in shifting environments or stresses. Yet, how these single-cell organisms vary and organize their behavior to tolerate stressors is poorly understood. This is because many bacterial subpopulations are rare and cannot be readily discovered by existing single-cell sequencing methods due to limitations in cell number and sequencing depth. Here we develop Massively-parallel Microbial mRNA sequencing (M3-Seq), which addresses these challenges by using combinatorially-indexed cells to overload droplets in combination with RNA amplification and post-hoc rRNA depletion. In a single M3-Seq experiment, we profile hundreds of thousands of bacterial cells from multiple species under a wide range of conditions. In addition to validating our approach and findings, we exploit the scale of M3-Seq to make several unexpected discoveries, including new insights into bet hedging strategies in stress responses, bacterial responses to antibiotics, and host responses to phage infection.
Competing Interest Statement
BA was a member of a ThinkLab Advisory Board for, and holds equity in, Celsius Therapeutics. ZG is the founder of ArrePath. The remaining authors declare no competing interests.
