Prokaryotic Single-Cell RNA Sequencing by In Situ Combinatorial Indexing

Abstract

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...

Spatial transcriptomics of planktonic and sessile bacterial populations at single-cell resolution

“…However, interpreting these maps remains challenging without additional functional information on the physiological states and activities of relevant community members. In contrast, recent adaptions of eukaryotic single-cell RNA-sequencing (scRNA-seq) approaches provide a powerful means of exploring the phenotypic landscape of planktonic bacteria (Blattman et al, 2020;Imdahl et al, 2020;Kuchina et al, 2021). However, these approaches do not preserve the spatial context of analyzed cells and are therefore limited in their capacity to address single and multispecies biofilms.…”

Prokaryotic single-cell RNA sequencing by in situ combinatorial indexing

“…Most notably, a customized tagmentation protocol during library preparation could theoretically increase the mRNA capture by 2-fold (see "Future directions for optimization" in Methods). Following our initial deposit of an earlier version of this manuscript on biorXiv 42 , and during its formal review, Kuchina and colleagues deposited a manuscript on biorXiv 43 reporting a conceptually similar split-pool based bacterial scRNA-seq method in which in situ polyadenylation was utilized to capture mRNAs. It will be of great interest to compare and contrast these methods and to further improve the performance of PETRI-seq.…”

Multicellular and unicellular responses of microbial biofilms to stress

“…This includes live single-cell microscopy techniques for biofilms (Hartmann et al 2019), and metabolite imaging techniques (Bellin et al 2016;Geier et al 2020). Furthermore, the rapidly improving techniques for bacterial single-cell RNA-seq measurements (Blattman et al 2020;Imdahl et al 2020;Kang et al 2011;Kuchina et al 2019) are very promising tools for studying the variation between cells, and whether the community behavior is different than the sum of all individual cell behaviors. Single-cell techniques can enable new paths for mechanistic studies of multicellular functions in biofilms.…”