PT  - JOURNAL ARTICLE
AU  - Bruce Wang
AU  - Aaron E. Lin
AU  - Jiayi Yuan
AU  - Matthias D. Koch
AU  - Britt Adamson
AU  - Ned S. Wingreen
AU  - Zemer Gitai
TI  - Massively-parallel Microbial mRNA Sequencing (M3-Seq) reveals heterogenous behaviors in bacteria at single-cell resolution
AID  - 10.1101/2022.09.21.508688
DP  - 2022 Jan 01
TA  - bioRxiv
PG  - 2022.09.21.508688
4099  - http://biorxiv.org/content/early/2022/09/22/2022.09.21.508688.short
4100  - http://biorxiv.org/content/early/2022/09/22/2022.09.21.508688.full
AB  - 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 StatementBA 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.