Despite longstanding appreciation of gene expression heterogeneity in isogenic bacterial populations, affordable and scalable technologies for studying single bacterial cells have been limited. Although single-cell RNA sequencing (scRNA-seq) has revolutionized studies of transcriptional heterogeneity in diverse eukaryotic systems^{1,2,3,4,5,6,7,8,9,10,11,12,13}, the application of scRNA-seq to prokaryotes has been hindered by their extremely low mRNA abundance^14,15,16, lack of mRNA polyadenylation and thick cell walls¹⁷. Here, we present prokaryotic expression profiling by tagging RNA in situ and sequencing (PETRI-seq)—a low-cost, high-throughput prokaryotic scRNA-seq pipeline that overcomes these technical obstacles. PETRI-seq uses in situ combinatorial indexing^11,12,18 to barcode transcripts from tens of thousands of cells in a single experiment. PETRI-seq captures single-cell transcriptomes of Gram-negative and Gram-positive bacteria with high purity and low bias, with median capture rates of more than 200 mRNAs per cell for exponentially growing Escherichia coli. These characteristics enable robust discrimination of cell states corresponding to different phases of growth. When applied to wild-type Staphylococcus aureus, PETRI-seq revealed a rare subpopulation of cells undergoing prophage induction. We anticipate that PETRI-seq will have broad utility in defining single-cell states and their dynamics in complex microbial communities.

尽管长期以来人们对同基因细菌群体中基因表达异质性的认识很高，但用于研究单个细菌细胞的经济且可扩展的技术仍然有限。尽管单细胞 RNA 测序 (scRNA-seq) 彻底改变了不同真核系统中转录异质性的研究^{1,2,3,4,5,6,7,8,9,10,11,12,13}，原核生物的 scRNA-seq 因其极低的 mRNA 丰度^14,15,16、缺乏 mRNA 多腺苷酸化和厚的细胞壁^{17 而}受到阻碍。在这里，我们提出了通过原位标记 RNA 和测序 (PETRI-seq) 进行原核表达谱分析，这是一种低成本、高通量的原核 scRNA-seq 流程，克服了这些技术障碍。PETRI-seq 在一次实验中使用原位组合索引^11、12、18对来自数万个细胞的条形码转录本进行标记。PETRI-seq 以高纯度和低偏差捕获革兰氏阴性和革兰氏阳性细菌的单细胞转录组，对于呈指数增长的大肠杆菌，每个细胞的中值捕获率超过 200 个 mRNA 。这些特征使得能够强有力地区分对应于不同生长阶段的细胞状态。当应用于野生型金黄色葡萄球菌时，PETRI-seq 揭示了正在经历原噬菌体诱导的罕见细胞亚群。我们预计 PETRI-seq 将在定义复杂微生物群落中的单细胞状态及其动态方面具有广泛的用途。