Profiling of bacterial transcriptome from ultra-low input with MiniBac-seq.

Area of Science:

• Microbiology
• Molecular Biology
• Genomics

Background:

• Standard bacterial RNA sequencing (RNA-seq) methods require substantial RNA input, limiting analysis of low-abundance populations.
• Ultra-low RNA amounts, such as from single cells or specific subpopulations, pose technical challenges for library preparation.
• Understanding the transcriptome of rare bacterial states is crucial for fields like antibiotic resistance and microbial physiology.

Purpose of the Study:

• To develop a sensitive and cost-effective method for bacterial RNA-seq library preparation from sub-nanogram total RNA.
• To enable high-quality transcriptome profiling of bacterial subpopulations with limited RNA availability.
• To investigate the gene expression of growth-arrested *Escherichia coli*, a reservoir of antibiotic persistence.

Main Methods:

• Development of miniBac-seq, a novel strand-specific RNA-seq library construction protocol.
• Optimization for ultra-low input RNA (sub-nanogram), achieving 100-fold reduction compared to benchmark kits.
• Application of miniBac-seq to profile the transcriptome of growth-arrested *E. coli*.

Main Results:

• miniBac-seq successfully generated high-quality libraries from sub-nanogram total RNA, significantly reducing costs.
• The method demonstrated high sensitivity, detecting over 500 genes from RNA equivalent to a single bacterial cell.
• Transcriptome profiling of growth-arrested *E. coli* identified potential molecular drivers of arrested growth and antibiotic tolerance.

Conclusions:

• miniBac-seq offers a powerful, cost-effective solution for bacterial RNA-seq with ultra-low RNA input.
• The method facilitates the study of previously inaccessible bacterial transcriptomes, including rare or small populations.
• This advancement aids in understanding bacterial physiology, regulation, and antibiotic tolerance in native contexts.