Jeff Dean (the Biochemist) @jeffdean@qoto.org

Bulk RNA-sequencing technologies have provided invaluable insights into host and bacterial gene expression and associated regulatory networks. Nevertheless, the majority of these approaches report average expression across cell populations, hiding the true underlying expression patterns that are often heterogeneous in nature. Due to technical advances, single-cell transcriptomics in bacteria has recently become reality, allowing exploration of these heterogeneous populations, which are often the result of environmental changes and stressors. In this work, we have improved our previously published bacterial single-cell RNA-sequencing protocol that is based on MATQ-seq, achieving a higher throughput through the integration of automation. We also selected a more efficient reverse transcriptase, which led to reduced cell loss and higher workflow robustness. Moreover, we successfully implemented a Cas9-based ribosomal RNA depletion protocol into the MATQ-seq workflow. Applying our improved protocol on a large set of single Salmonella cells sampled over growth revealed improved gene coverage and a higher gene detection limit compared to our original protocol and allowed us to detect the expression of small regulatory RNAs, such as GcvB or CsrB at a single-cell level. In addition, we confirmed previously described phenotypic heterogeneity in Salmonella in regards to expression of pathogenicity-associated genes. Overall, the low percentage of cell loss and high gene detection limit makes the improved MATQ-seq protocol particularly well suited for studies with limited input material, such as analysis of small bacterial populations in host niches or intracellular bacteria. IMPORTANCE Gene expression heterogeneity among isogenic bacteria is linked to clinically-relevant scenarios, like biofilm formation and antibiotic tolerance. The recent development of bacterial single-cell RNA-sequencing (scRNA-seq) enables the study of cell-to-cell variability in bacterial populations and the mechanisms underlying these phenomena. Here, we report a scRNA-seq workflow based on MATQ-seq with increased robustness, reduced cell loss, improved transcript capture rate, and gene coverage. Use of a more efficient reverse transcriptase and the integration of a ribosomal RNA depletion step, which can be adapted to other bacterial single-cell workflows, was instrumental for these improvements. Applying the protocol to the foodborne-pathogen Salmonella , we confirmed transcriptional heterogeneity across and within different growth phases and demonstrated that our workflow captures small regulatory RNAs on the single-cell level. Due to low cell loss and high transcript capture rates, this protocol is uniquely suited for experimental settings in which the starting material is limited, such as infected tissues. ### Competing Interest Statement The authors have declared no competing interest.

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