Fig. 3: M3-seq enables systematic investigation of bacterial response to antibiotic treatment.

a, Schematics of antibiotic experiment (eBW4). During preparation of M3-seq gene expression libraries, round-one plate indexing was used to uniquely mark antibiotic-treated and untreated cultures. Plate and tubes are adapted from BioRender.com. b, Heat map depicts Pearson correlations of pseudobulk transcriptomes from E. coli MG1655 prepared as in a, which were computed using genes with average expression greater than the median average expression of all genes. Coloured text indicates antibiotics of similar mechanisms of action. c, Same as b but for B. subtilis 168. d, UMAP of E. coli MG1655 transcriptomes from cells treated with the bacteriostatic antibiotics tetracycline and chloramphenicol. Colours indicate drug treatment. e, Same as d but with colours indicating clusters of transcriptionally similar cells. Percentage of cells in each cluster is denoted. f, Same as d but with colour gradient indicating the normalized UMI count of MGEs. Clusters 8, 12, 13 and 16 were enriched for MGE expression. g, Cell rarefaction analyses using M3-seq data. Curves indicate kurtosis of 15 principal components computed from tetracycline- and chloramphenicol-treated E. coli MG1655 cells, with individual curves corresponding to calculations from the total population of cells (79,804) or downsampled populations thereof (down to 1,000 cells). The 15 principal components included were those with the highest kurtosis for each downsampling. Inset UMAPs were computed from each downsampled data matrix. Within the embeddings, magenta indicates members of cluster 16 (indicated in f), which can only be distinguished for >7,500–10,000 cells. Notably, the top row of embeddings (2,500–10,000 cells) represents the scale of experiments from previous studies, while the bottom row represents the scale enabled by M3-seq. h, UMI rarefaction experiments using M3-seq data. Curves indicate kurtosis of 15 principal components computed from 79,804 tetracycline- and chloramphenicol-treated E. coli MG1655 cells, with individual curves corresponding to data subsampled for UMIs per cell (7 to 56 median UMIs). The 15 principal components included were those with the highest kurtosis for each subsampling of UMIs. Inset UMAPs were computed from each subsampled data matrix. Within the embeddings, magenta indicates members of cluster 16 (indicated in f), which can only be distinguished at the highest UMI detection efficiency.