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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. Although single-cell RNA sequencing (scRNA-seq) has revolutionized studies of transcriptional heterogeneity in diverse eukaryotic systems^1-13, the application of scRNA-seq to prokaryotes has been hindered by their extremely low mRNA abundance^14-16, lack of mRNA polyadenylation and thick cell walls¹⁷. 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 of more than 200 mRNAs per cell for exponentially growing Escherichia coli. These characteristics enable robust discrimination of cell states corresponding to different phases of growth. When applied to wild-type Staphylococcus aureus, PETRI-seq revealed a rare subpopulation of cells undergoing prophage induction. We anticipate that PETRI-seq will have broad utility in defining single-cell states and their dynamics in complex microbial communities.

SUBMITTER: Blattman SB

PROVIDER: S-EPMC8330242 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Prokaryotic Single-Cell RNA Sequencing by In Situ Combinatorial Indexing

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