show Abstracthide AbstractWe introduce microSPLiT, a low cost and high-throughput single-cell RNA-sequencing method for gram-negative and gram-positive bacteria. We applied microSPLiT to >25,000 Bacillus subtilis cells sampled at different growth stages, creating a detailed atlas of changes in metabolism and lifestyle. We not only retrieve detailed gene expression profiles associated with known, but rare, states such as competence and PBSX prophage induction, but also identify novel and unexpected gene expression states including a heterogeneous activation of a niche metabolic pathway (myo-inositol catabolism) in a subpopulation of cells. Overall design: Single-cell RNA-seq (microSPLiT) was used to sample ten optical density (OD) points along the growth curve of the laboratory strain PY79 in LB medium ranging from OD0.5 to 6.0, with one replicate of 4 OD points. For validation of the method, we grew E. coli MW1255 and B. subtilis PY79 cells to OD600=0.5 and subjected half of each culture to a brief 47°C heat-shock. We performed a microSPLiT experiment on both samples, using the first barcode as a sample identifier.