A Microcapsule-Integrated smRandom-seq Platform Enables Fixation-Free Single Microbe RNA Sequencing

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• Current single-microbe RNA sequencing (smRNA-seq) relies on chemical fixation, potentially impacting results.
• A practical, fixation-free alternative is needed to fully understand smRNA-seq performance.

Purpose of the Study:

• To develop and validate a fixation-free smRNA-seq workflow using microcapsule technology.
• To assess the impact of fixation-free processing on transcriptomic data quality and sensitivity.

Main Methods:

• Integration of aqueous two-phase system microcapsules with the smRandom-seq method.
• Physical confinement of bacterial cells and nucleic acids within microcompartments.
• Application to Escherichia coli and Acinetobacter baumannii for fixation-based and fixation-free workflows.

Main Results:

• The microcapsule-integrated smRNA-seq platform enables fixation-free single-cell transcript capture.
• Fixation-free processing increased per-cell gene detection sensitivity by ~50% compared to fixed controls.
• Overall transcriptional profiles were preserved similarly between fixed and fixation-free methods.

Conclusions:

• The developed platform offers a practical alternative to chemical fixation in high-throughput smRNA-seq.
• This fixation-free approach significantly enhances transcriptomic capture sensitivity at the single-cell level.
• The technology advances single-microbe omics and microbial heterogeneity studies.