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

Ziye Xu; Yuting Wang; Kuanwei Sheng; Raoul 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

doi:10.1038/s41467-023-40137-9

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

Nat Commun. 2023 Aug 23;14(1):5130. doi: 10.1038/s41467-023-40137-9.

Authors

Ziye Xu^#^{1

2}, Yuting Wang^#^{2

3}, Kuanwei Sheng^#^{4

5}, Raoul Rosenthal⁶, Nan Liu², Xiaoting Hua⁷, Tianyu Zhang², Jiaye Chen⁸, Mengdi Song², Yuexiao Lv², Shunji Zhang³, Yingjuan Huang², Zhaolun Wang², Ting Cao^{1

9

6}, Yifei Shen¹, Yan Jiang⁷, Yunsong Yu⁷, Yu Chen¹, Guoji Guo², Peng Yin^{10

11}, David A Weitz^{12

13}, Yongcheng Wang^{14

15

16

17}

Affiliations

¹ Department of Laboratory Medicine, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.
² Liangzhu Laboratory, Zhejiang University, Hangzhou, China.
³ College of Biomedical Engineering and Instrument Science, Zhejiang University, Hangzhou, China.
⁴ Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, USA. Kuanwei.Sheng@wyss.harvard.edu.
⁵ Department of Systems Biology, Harvard Medical School, Boston, MA, USA. Kuanwei.Sheng@wyss.harvard.edu.
⁶ John A. Paulson School of Engineering and Applied Sciences and Department of Physics, Harvard University, Cambridge, MA, USA.
⁷ Department of Infectious Diseases, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China.
⁸ Department of Biomedical Informatics, Harvard Medical School, Boston, MA, USA.
⁹ Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, USA.
¹⁰ Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, USA. Peng_Yin@hms.harvard.edu.
¹¹ Department of Systems Biology, Harvard Medical School, Boston, MA, USA. Peng_Yin@hms.harvard.edu.
¹² Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, USA. weitz@seas.harvard.edu.
¹³ John A. Paulson School of Engineering and Applied Sciences and Department of Physics, Harvard University, Cambridge, MA, USA. weitz@seas.harvard.edu.
¹⁴ Department of Laboratory Medicine, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China. yongcheng@zju.edu.cn.
¹⁵ Liangzhu Laboratory, Zhejiang University, Hangzhou, China. yongcheng@zju.edu.cn.
¹⁶ College of Biomedical Engineering and Instrument Science, Zhejiang University, Hangzhou, China. yongcheng@zju.edu.cn.
¹⁷ Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, USA. yongcheng@zju.edu.cn.

^# Contributed equally.

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.

Publication types

Research Support, U.S. Gov't, Non-P.H.S.
Research Support, Non-U.S. Gov't

MeSH terms

Anti-Bacterial Agents / pharmacology
DNA Primers
Escherichia coli* / genetics
High-Throughput Nucleotide Sequencing*
Humans
RNA, Ribosomal / genetics
RNA-Seq

Substances

Anti-Bacterial Agents
DNA Primers
RNA, Ribosomal