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

• Published in: Nature communications Vol. 14; no. 1; p. 5130
• Main Authors: Xu, Ziye, Wang, Yuting, Sheng, Kuanwei, Rosenthal, Raoul, Liu, Nan, Hua, Xiaoting, Zhang, Tianyu, Chen, Jiaye, Song, Mengdi, Lv, Yuexiao, Zhang, Shunji, Huang, Yingjuan, Wang, Zhaolun, Cao, Ting, Shen, Yifei, Jiang, Yan, Yu, Yunsong, Chen, Yu, Guo, Guoji, Yin, Peng, Weitz, David A., Wang, Yongcheng
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 23.08.2023; Nature Publishing Group; Nature Portfolio
• Subjects: 38; 38/62; 38/91; 631/1647/2234; 631/1647/350/877; 631/208/514/1949; Antibiotic resistance; Antibiotics; Bacteria; Bar codes; CRISPR; Depletion; Droplets; E coli; Gene expression; Gene sequencing; Heterogeneity; Humanities and Social Sciences; Metabolic pathways; Microbiomes; Microorganisms; multidisciplinary; Ribonucleic acid; RNA; rRNA; Science; Science (multidisciplinary); SOS response; Subpopulations; Transcriptomes; Transcriptomics
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-023-40137-9

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.