Droplet-based high-throughput single microbe RNA sequencing by smRandom-seq

Area of Science:

• Microbiology
• Genomics
• Molecular Biology

Background:

• Bacteria inhabit diverse environments, including the human body, with significant individual variations.
• Population-level transcriptomics masks crucial heterogeneity within bacterial communities.
• Understanding single-bacterial gene expression is vital for fields like antimicrobial resistance and microbiome research.

Purpose of the Study:

• To develop a high-throughput, droplet-based assay for single-microbe RNA sequencing (smRandom-seq).
• To enable detailed analysis of transcriptome heterogeneity in bacterial populations.
• To identify specific gene expression patterns associated with microbial traits like antibiotic resistance.

Main Methods:

• Droplet-based microfluidics for single-microbe encapsulation and barcoding.
• Random primers for in situ cDNA synthesis within droplets.
• CRISPR-based ribosomal RNA (rRNA) depletion for enhanced mRNA enrichment.
• High-throughput single-microbe RNA sequencing (smRandom-seq) analysis.

Main Results:

• smRandom-seq demonstrated high species specificity (>99%) and a low doublet rate (1.6%).
• The method achieved reduced rRNA contamination (32%) and sensitive gene detection (median ~1000 genes/E. coli).
• Transcriptome analysis of individual E. coli revealed distinct subpopulations with unique SOS response and metabolic pathway gene expression under antibiotic stress.

Conclusions:

• smRandom-seq is a powerful tool for high-throughput single-microbe transcriptome profiling.
• This technology facilitates the study of microbial resistance, persistence, and microbe-host interactions.
• The findings open new avenues for microbiome research by dissecting population heterogeneity.