Prokaryotic Single-Cell RNA Sequencing by In Situ Combinatorial Indexing

We present Prokaryotic Expression-profiling by Tagging RNA In Situ and sequencing (PETRI-seq), a high-throughput prokaryotic scRNA-seq pipeline. We demonstrated that PETRI-seq effectively barcoded single bacterial cells in a species-mixing experiment with E. coli (MG1655) and S. aureus (USA300). Within the S. aureus population, we found rare prophage induction in 0.04% of cells. We further demonstrated that PETRI-seq was able to distinguish between E. coli growth phases based on mRNA expression patterns by combining stationary E. coli with exponential E. coli in multiple experiments. Eight libraries were prepared by PETRI-seq, including species-mixed libraries of E. coli and S. aureus and/or E. coli cells in distinct growth stages (stationary, exponential). PETRI-seq libraries are detailed in Supplementary Table 4. Ten bulk libraries were prepared, including nine E. coli libraries and one S. aureus library.