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 1 – 13 , application of scRNA-seq to prokaryotes has been hindered by their extremely low mRNA abundance 14 – 16 , lack of mRNA polyadenylation, and thick cell walls 17 . 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 11 , 12 , 18 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 >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.

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 >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. MainRecent developments in high-throughput single-cell RNA sequencing (scRNA-seq) technology have enabled rapid characterization of cellular diversity within complex eukaryotic tissues 1-13 . Despite these advances, comparable tools to study the transcriptomes of individual bacterial cells 14-16 have lagged behind significantly due to numerous technical challenges (Fig. S1). Current massively parallel eukaryotic scRNA-seq methods typically require custom microfluidics to co-encapsulate a single cell with a uniquely barcoded bead in a compartment, often a droplet 5,6,8 or microwell 4,7 . These approaches rely on two key properties of many eukaryotic cells, specifically that they are easily lysed with detergent to release their RNA and that their polyadenylated mRNAs can be effectively captured by beads coated with poly(dT) primers. Adaptation of these approaches for bacteria is thwarted by the presence of thick prokaryotic cell wall 17 , which makes lysis challenging, and the lack of poly-adenylated mRNAs for effective capture.Given these considerations, we identified in situ combinatorial indexing 18 as an alternative basis upon which to develop a method for high-throughput prokaryotic scRNA-seq. Two conceptually similar eukaryotic methods, single-cell combinatorial indexing RNA sequencing (sci-RNA-seq) 11,13 and split-pool ligation-based transcriptome sequencing (SPLiT-seq) 12 , rely on cells themselves as compartments for barcoding, which abrogates the need for cell lysis in droplets or microwells. These methods are also amenable to reverse transcription (RT) with random hexamers instead of poly(dT) primers 12 . With just pipetting steps and no complex instruments, individual t...

The α7-nicotinic acetylcholine receptor (α7-nAChR) is a ligand-gated ion channel widely expressed in vertebrates and is associated with numerous physiological functions. As transmembrane ion channels, α7-nAChRs need to be expressed on the surface of the plasma membrane to function. The receptor has been reported to associate with proteins involved with receptor biogenesis, modulation of receptor properties, as well as intracellular signaling cascades and some of these associated proteins may affect surface expression of α7-nAChRs. The putative chaperone resistance to inhibitors of cholinesterase 3 (Ric-3) has been reported to interact with, and enhance the surface expression of, α7-nAChRs. In this study, we identified proteins that associate with α7-nAChRs when Ric-3 is expressed. Using α-bungarotoxin (α-bgtx), we isolated and compared α7-nAChR-associated proteins from two stably transfected, human tumor-derived cell lines: SH-EP1-hα7 expressing human α7-nAChRs and the same cell line further transfected to express Ric-3, SH-EP1-hα7-Ric-3. Mass spectrometric analysis of peptides identified thirty-nine proteins that are associated with α7-nAChRs only when Ric-3 was expressed. Significantly, and consistent with reports of Ric-3 function in the literature, several of the identified proteins are involved in biological processes that may affect nAChR surface expression such as post-translational processing of proteins, protein trafficking, and protein transport. Additionally, proteins affecting the cell cycle, the cytoskeleton, stress responses, as well as cyclic AMP- and inositol triphosphate-dependent signaling cascades were identified. These results illuminate how α-bgtx may be used to isolate and identify α7-nAChRs as well as how the expression of chaperones such as Ric-3 can influence proteins associating with α7-nAChRs. These associating proteins may alter activities of α7-nAChRs to expand their functionally-relevant repertoire as well as to affect biogenesis and membrane trafficking of α7-nAChRs.