High-throughput single-cell transcriptomics of bacteria using combinatorial barcoding.

Area of Science:

• Microbiology
• Genomics
• Molecular Biology

Background:

• Single-cell RNA sequencing (scRNA-seq) is crucial for understanding cellular heterogeneity.
• Existing bacterial scRNA-seq methods often require specialized equipment or are limited in scale.

Purpose of the Study:

• To introduce microbial split-pool ligation transcriptomics (microSPLiT), a high-throughput scRNA-seq method for bacteria.
• To enable scalable transcriptional profiling of hundreds of thousands to millions of bacterial cells.

Main Methods:

• microSPLiT utilizes four rounds of combinatorial barcoding for transcript labeling within fixed and permeabilized bacterial cells.
• In-cell ligation reactions append barcodes after reverse transcription.
• Samples can be stored after fixation and initial barcoding rounds.

Main Results:

• microSPLiT can profile transcriptional states in hundreds of thousands of Gram-negative and Gram-positive bacteria in a single experiment.
• The protocol generates sequencing-ready libraries in 4 days.
• Standard setup supports up to 1 million bacterial cells and 96 samples, with scalability.

Conclusions:

• microSPLiT provides a cost-effective and scalable solution for bacterial single-cell transcriptomics.
• The method is accessible to researchers with basic molecular biology experience.
• Bacterial samples can be collected and stored, allowing flexibility in experimental design.