PT - JOURNAL ARTICLE AU - Sydney B. Blattman AU - Wenyan Jiang AU - Panos Oikonomou AU - Saeed Tavazoie TI - Prokaryotic Single-Cell RNA Sequencing by <em>In Situ</em> Combinatorial Indexing AID - 10.1101/866244 DP - 2020 Jan 01 TA - bioRxiv PG - 866244 4099 - http://biorxiv.org/content/early/2020/03/18/866244.short 4100 - http://biorxiv.org/content/early/2020/03/18/866244.full AB - Despite longstanding appreciation of gene expression heterogeneity in isogenic bacterial populations, affordable and scalable technologies for studying single bacterial cells have been limited. While single-cell RNA sequencing (scRNA-seq) has revolutionized studies of transcriptional heterogeneity in diverse eukaryotic systems, application of scRNA-seq to prokaryotes has been hindered by their extremely low mRNA abundance, lack of mRNA polyadenylation, and thick cell walls. Here, we present Prokaryotic Expression-profiling by Tagging RNA In Situ and sequencing (PETRI-seq), a low-cost, high-throughput, prokaryotic scRNA-seq pipeline that overcomes these technical obstacles. PETRI-seq uses in situ combinatorial indexing to barcode transcripts from tens of thousands of cells in a single experiment. PETRI-seq captures single cell transcriptomes of Gram-negative and Gram-positive bacteria with high purity and low bias, with median capture rates &gt;200 mRNAs/cell for exponentially growing E. coli. These characteristics enable robust discrimination of cell-states corresponding to different phases of growth. When applied to wild-type S. aureus, PETRI-seq revealed a rare sub-population of cells undergoing prophage induction. We anticipate broad utility of PETRI-seq in defining single-cell states and their dynamics in complex microbial communities.