Improved bacterial single-cell RNA-seq through automated MATQ-seq [bulk RNA-seq]

In this work, we have improved our previously published bacterial single-cell RNA-sequencing protocol (MATQ-seq), providing enhancements that achieve a higher cell throughput while also including integration of automation. We selected a more efficient reverse transcriptase which led to a lower drop-out rate and higher workflow robustness, and we also successfully implemented a Cas9-based ribosomal RNA depletion protocol into the MATQ-seq workflow. Applying this improved protocol on a large set of single Salmonella cells sampled over growth revealed improved gene coverage and a higher gene detection limit, allowing us to reveal the expression of small regulatory RNAs such as GcvB or CsrB at a single-cell level. In addition, we were able to confirm previously described phenotypic heterogeneity in Salmonella in regards to expression of pathogenicity-associated genes. Overall design: Bulk RNA-seq matching four growth phases used in parallel with single cell sequencing from Salmonella enterica serovar Typhimurium.