ProBac-seq, a bacterial single-cell RNA sequencing methodology using droplet microfluidics and large oligonucleotide...

Area of Science:

• Microbiology
• Molecular Biology
• Genomics

Background:

• Single-cell transcriptomics has revolutionized eukaryotic cell studies.
• Existing methods are unsuitable for bacteria due to RNA instability and cell morphology.
• Bacterial RNA lacks polyadenylation, posing a challenge for transcriptomic analysis.

Purpose of the Study:

• To present ProBac sequencing (ProBac-seq), a novel method for bacterial single-cell transcriptomics.
• To detail the protocol for designing, preparing, and applying ProBac-seq.
• To enable high-resolution gene expression analysis in individual bacterial cells.

Main Methods:

• Design and amplification of custom mRNA-specific oligonucleotide probe sets.
• Addition of unique molecular indexes and poly-A tails to probes.
• In situ probe hybridization, single-cell encapsulation, and library preparation.

Main Results:

• ProBac-seq yields high-quality single-cell gene expression data from thousands of bacterial cells.
• The method effectively captures target mRNA, avoiding abundant ribosomal RNA.
• ProBac-seq allows detection of low-level transcript signals and is compatible with various organisms.

Conclusions:

• ProBac-seq overcomes key technical challenges for bacterial single-cell transcriptomics.
• This method enhances understanding of bacterial cellular heterogeneity and gene expression.
• ProBac-seq is a versatile tool for studying bacteria in diverse, environmentally relevant conditions.