SRP492308 PRJNA1081799 GSE260457 An Improved Bacterial Single-cell RNA-seq Reveals Biofilm Heterogeneity [scRNA-seq] In contrast to mammalian cells, bacterial cells lack mRNA polyadenylated tails, presenting a hurdle in isolating mRNA amidst the prevalent rRNA during single-cell RNA-seq. This study introduces a novel method, Ribosomal RNA-derived cDNA Depletion (RiboD), seamlessly integrated into the PETRI-seq technique, yielding RiboD-PETRI. This innovative approach offers a cost-effective, equipment-free, and high-throughput solution for bacterial single-cell RNA sequencing (scRNA-seq). By efficiently eliminating rRNA reads and substantially enhancing mRNA detection rates (up to 92%), our method enables precise exploration of bacterial population heterogeneity. Applying RiboD-PETRI to investigate biofilm heterogeneity, distinctive subpopulations marked by unique genes within biofilms were successfully identified. Notably, PdeI, a marker for the cell-surface attachment subpopulation, was observed to elevate cyclic diguanylate (c-di-GMP) levels, promoting persister cell formation. Thus, we address a persistent challenge in bacterial single-cell RNA-seq regarding rRNA abundance, exemplifying the utility of this method in exploring biofilm heterogeneity. Our method effectively tackles a long-standing issue in bacterial scRNA-seq: the overwhelming abundance of rRNA. This advancement significantly enhances our ability to investigate the intricate heterogeneity within biofilms at unprecedented resolution. Overall design: We performed a new single-cell library construction method for three species of bacteria: Escherichia coli MG1655, Caulobacter crescentus strain NA1000, Staphylococcus aureus 25923 and verified the effect of different treatments during library construction on library data. And then we analyzed the single-cell sequencing datas by Seurat(version 4.3.0; http://satijalab.org/seurat/). GSE260457 parent_bioproject PRJNA1081803