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

## TL;DR

This paper introduces smRandom-seq, a new method for studying gene activity in individual bacteria, revealing hidden differences in bacterial populations.

## Contribution

The novel smRandom-seq method enables high-throughput single-microbe RNA sequencing with high specificity and sensitivity.

## Key findings

- smRandom-seq achieves 99% species specificity and detects ~1000 genes per single E. coli cell.
- The method successfully identifies antibiotic-resistant subpopulations with distinct gene expression patterns.
- CRISPR-based rRNA depletion reduces rRNA percentage to 32%, improving mRNA detection.

## Abstract

Bacteria colonize almost all parts of the human body and can differ significantly. However, the population level transcriptomics measurements can only describe the average bacteria population behaviors, ignoring the heterogeneity among bacteria. Here, we report a droplet-based high-throughput single-microbe RNA-seq assay (smRandom-seq), using random primers for in situ cDNA generation, droplets for single-microbe barcoding, and CRISPR-based rRNA depletion for mRNA enrichment. smRandom-seq showed a high species specificity (99%), a minor doublet rate (1.6%), a reduced rRNA percentage (32%), and a sensitive gene detection (a median of ~1000 genes per single E. coli). Furthermore, smRandom-seq successfully captured transcriptome changes of thousands of individual E. coli and discovered a few antibiotic resistant subpopulations displaying distinct gene expression patterns of SOS response and metabolic pathways in E. coli population upon antibiotic stress. smRandom-seq provides a high-throughput single-microbe transcriptome profiling tool that will facilitate future discoveries in microbial resistance, persistence, microbe-host interaction, and microbiome research.

Population level transcriptomics measurements miss bacterial heterogeneity. Here the authors report smRandom-seq, a droplet-based high-throughput single-microbe RNA-seq assay, using random primers for in situ cDNA generation, droplets for single-microbe barcoding, and CRISPR-based rRNA depletion.