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, the application of scRNA-seq to prokaryotes has been hindered by their extremely low mRNA abundance, lack of mRNA polyadenylation and thick cell walls.
Columbia University researchers have developed 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 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. The researchers anticipate that PETRI-seq will have broad utility in defining single-cell states and their dynamics in complex microbial communities.
Overview of PETRI-seq
PETRI-seq includes three parts—cell preparation, split-pool barcoding and library preparation. During cell preparation, cells are prepared for in situ reactions by fixation (formaldehyde) and permeabilization (lysozyme/lysostaphin). During split-pool barcoding, cells are split across 96-well plates three times for three rounds of barcoding by RT and two ligations. After barcoding, cells are lysed to release cDNA, which is subsequently prepared for paired-end Illumina sequencing. Each cDNA fragment in the library includes a UMI and three barcodes, which are all sequenced in read 1. The UMI is a sequence of seven degenerate nucleotides that can distinguish between unique transcripts and PCR duplicates. The three barcodes comprise a BC, which enables reads to be grouped by their cell of origin. In read 2, the cDNA is sequenced.
Availability – current PETRI-seq code and protocols are available at https://tavazoielab.c2b2.columbia.edu/PETRI-seq/.