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. Overall design: 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.