Microbial transcriptomes are commonly small, with only a few copies of each transcript due to their lack of alternative splicing mechanism. The mRNAs of prokaryotes usually lack polyadenylation (polyA); therefore, traditional eukaryotic single-cell RNA-seq methods relying on polyA tailing protocols are not suitable for prokaryotic transcriptome analysis. Additionally, cell walls of microbes are usually tough, composed of beta-glucan, chitin, and manno-protein. These characteristics of microorganisms make microbial single-cell RNA-seq a challenging task. To solve this problem, we developed and validated a robust microbial single-cell RNA sequencing platform.

Fluorescence-activated cell sorting (FACS), micromanipulation or microfluidic platform are used to harvest single cells. After RNA isolation and quality control, extracted RNAs are reversely transcribed and amplified, and used to construct libraries using improved Smart-seq method or commercial kits. Validated libraries are then subjected to Illumina sequencing (PE100/SE50). Sequenced reads are preprocessed and analyzed by our experienced bioinformatics experts. We help to investigate the gene expression heterogeneity among individual cells, which can be a major determinant of the success in antimicrobial treatment and disease outcome.