Droplet-based high-throughput single microbe RNA sequencing by smRandom-seq
Ziye Xu, Yuting Wang, Kuanwei Sheng, Raoul G Rosenthal, Nan Liu, Xiaoting Hua, Tianyu Zhang, Jiaye Chen, Mengdi Song, Yuexiao Lv, Shunji Zhang, Yingjuan Huang, Zhaolun Wang, Ting Cao, Yifei Shen, Yan Jiang, Yunsong Yu, Yu Chen, Guoji Guo, Peng Yin, David A. Weitz, Yongcheng Wang, Yongcheng Wang, Yongcheng Wang
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.