Highly multiplexed spatial transcriptomics in bacteria

Single-cell decisions made in complex environments underlie many bacterial phenomena. Image-based, transcriptomics approaches offer an avenue to study such behaviors, yet these approaches have been hindered by the massive density of bacterial mRNA. To overcome this challenge, we combine 1000-fold volumetric expansion with multiplexed error robust fluorescence in situ hybridization (MERFISH) to create bacterial-MERFISH, a method enabling high-throughput, spatially resolved profiling of thousands of operons within individual bacteria. Using bacterial-MERFISH, we dissect the response of E. coli to carbon starvation, systematically map subcellular RNA organization, and chart the adaptation of B. thetaiotaomicron to micron-scale niches in the mammalian colon. We envision bacterial-MERFISH could prove useful in the study of bacterial single-cell decisions made in diverse, spatially structured, and native environments. Overall design: To measure and validate the performance of bacterial-MERFISH in Escherichia coli (E. coli), we correlated our MERFISH data with bulk RNA-seq of matching samples. We grew and collected WT E. coli MG1655 at OD600=0.33 in Lennox LB, and in diauxic media (0.025% w/v glucose and 0.025% w/v xylose in MOPS minimal media) at OD600=0.18 in the glucose growth phase, and 0.47 during xylose growth. We extracted total RNA following the RNAsnap protocol (Stead et al. 2012). The total RNA was ribosomally depleted and sequenced as 150bp paired-end reads by the Harvard Medical School Biopolymers Core (diauxic samples) and Azenta (LB sample).