mDrop-Seq: Massively Parallel Single-Cell RNA-Seq of and .

Advances in high-throughput single-cell RNA sequencing (scRNA-seq) have been limited by technical challenges such as tough cell walls and low RNA quantity that prevent transcriptomic profiling of microbial species at throughput. We present microbial Drop-seq or mDrop-seq, a high-throughput scRNA-seq technique that is demonstrated on two yeast species, , a popular model organism, and , a common opportunistic pathogen. We benchmarked mDrop-seq for sensitivity and specificity and used it to profile 35,109 cells to detect variation in mRNA levels between them. As a proof of concept, we quantified expression differences in heat shock using mDrop-seq. We detected differential activation of stress response genes within a seemingly homogenous population of under heat shock. We also applied mDrop-seq to cells, a polymorphic and clinically relevant species of yeast with a thicker cell wall compared to . Single-cell transcriptomes in 39,705 cells were characterized using mDrop-seq under different conditions, including exposure to fluconazole, a common anti-fungal drug. We noted differential regulation in stress response and drug target pathways between cells, changes in cell cycle patterns and marked increases in histone activity when treated with fluconazole. We demonstrate mDrop-seq to be an affordable and scalable technique that can quantify the variability in gene expression in different yeast species. We hope that mDrop-seq will lead to a better understanding of genetic variation in pathogens in response to stimuli and find immediate applications in investigating drug resistance, infection outcome and developing new drugs and treatment strategies.