![]() ![]() This approach requires generating large oligonucleotide libraries that are complementary to the protein-coding sequences within a genome (Supplementary Fig. To leverage existing microfluidic single-cell sequencing platforms, we devised a method whereby individual transcripts are tagged with DNA probes. Here we present a method called probe-based bacterial sequencing (ProBac-seq) for prokaryotic scRNA-seq, which uses a commercial, benchtop microfluidic device (Chromium Controller from 10X Genomics) and custom single-stranded DNA probe libraries to resolve the mRNA profile of thousands of bacterial cells. We reasoned that a method combining the advantages of microfluidic single-cell barcoding in droplets with the ability to tag transcripts using in situ hybridization of oligonucleotide probes could overcome these challenges and offer advantages over alternative approaches 5, 6, 7. Lastly, accessing mRNA requires cell permeabilization, which is difficult in bacteria due to the diversity in membrane structures and variability of the peptidoglycan layer. ![]() ![]() Third, bacterial transcripts do not intrinsically include a 3′ poly-adenosine tail and, therefore, mRNA cannot easily be tagged and selectively enriched against ribosomal RNA, which makes up more than 90% of the bacterial transcriptome 4. Second, transcriptional turnover is much faster in bacteria, with an mRNA half-life on the scale of minutes, compared with hours in eukaryotes 4. First, total bacterial messenger RNA (mRNA) abundance is two orders of magnitude lower than that of eukaryotes, with a single bacterial cell containing approximately 10 3–10 4 transcripts during exponential growth 4. However, tools for single-cell RNA sequencing (scRNA-seq) of bacterial populations remain limited due to several substantial technical challenges. Overall, ProBac-seq can be used to uncover heterogeneity in isogenic microbial populations and identify perturbations that affect pathogenicity.īacterial traits such as competence, sporulation and motility have been shown to be both heterogeneously utilized in populations and tightly controlled at the transcriptional level, emphasizing the need for single-cell transcriptional analyses 1, 2, 3. In the context of bacterial pathogenesis, application of the approach to Clostridium perfringens reveals heterogeneous expression of toxin by a subpopulation that can be controlled by acetate, a short-chain fatty acid highly prevalent in the gut. Applied to Bacillus subtilis and Escherichia coli, ProBac-seq correctly identifies known cell states and uncovers previously unreported transcriptional heterogeneity. We sequenced the transcriptome of thousands of individual bacterial cells per experiment, detecting several hundred transcripts per cell on average. Here we developed probe-based bacterial sequencing (ProBac-seq), a method that uses libraries of DNA probes and an existing commercial microfluidic platform to conduct bacterial single-cell RNA sequencing. Understanding all cell states requires studying isogenic bacterial populations at the single-cell level. Clonal bacterial populations rely on transcriptional variation across individual cells to produce specialized states that increase fitness. ![]()
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