Improving Single-Cell RNA Sequencing in Bacteria with Automation and Cas9

[seqadmin]

While bulk RNA sequencing has been vital for increasing our understanding of transcriptomics, single-cell RNA sequencing (scRNA-seq) has further enhanced these insights and provided a higher resolution of expression patterns by examining individual cells. This method has not only assisted with research in eukaryotes, but also with microorganisms like bacteria that are often found in heterogeneous populations.

Researchers from the Julius-Maximilians-Universität Würzburg (JMU) that previously developed a scRNA-seq protocol in bacteria have made significant improvements to their work, which were recently reported in the journal mBio. The older protocol was based on multiple annealing and deoxycytidine (dC) tailing-based quantitative scRNA-seq (MATQ-seq) adapted for bacteria.

Enhanced protocol
The first improvement to the procedure came from the integration of automation. This allowed the researchers to achieve a higher cell throughput and increase the technique's robustness by cutting out labor-intensive pipetting steps, leading to a reduced failure rate and improved quality control. In addition, the data processing and analysis pipelines were updated to increase data quality. The team implemented a better trimming approach, alignment, normalization, and identification of outliers.

Further refinements to the protocol were facilitated by using a more efficient reverse transcriptase. The newer transcriptase had improved processivity, robustness, and thermostability, which reduced cell loss and maintained higher reproducibility.

Moreover, the group was able to reduce rRNA reads with a Cas9-mediated cleavage protocol called DASH. Using a pool of single guide RNAs (sgRNAs), they targeted rRNA-derived cDNA for Cas9 cleavage, resulting in an increased rRNA depletion efficiency.

Testing the changes
To test this improved protocol, the team sampled individual Salmonella cells across different growth conditions to understand the changes to throughput and robustness. The results show an increase in gene coverage and gene deception limit, as well as the ability to detect small regulatory RNAs (sRNA). Identifying sRNAs like GcvB or CsrB at the single-cell level was not previously possible using the older protocol.

The results also confirmed previous work on heterogeneity in Salmonella highlighting the expression of pathogenicity-associated genes. The researchers believe these results make the improved protocol appropriate for sequencing experiments with limited input material, particularly small bacterial populations in host niches or with intracellular bacteria.