scRNA of Streptococcus pneumoniae
Time-kill assays were performed for S. pneumoniae WT and rnyL77S under cefepime exposure (0.064 g/mL) and recovery, with samples collected at 0, 60, 120, and 240 min during exposure and at 120 and 240 min post-treatment. Additional rnyL77S cultures were exposed to levofloxacin, vancomycin, high osmolarity (300 mM NaCl), or acidic pH (4.2) for comparison. Cell numbers were normalized by optical density, fixed with 1% paraformaldehyde, and permeabilized with phage-derived CPL1 enzyme. After in situ probe hybridization, single cells were encapsulated and barcoded using the 10x Genomics Chromium Next GEM Single Cell 3 Kit v3.1. Libraries were prepared according to the manufacturers instructions, validated by TapeStation, and sequenced on an AVITI 2150 bp Cloudbreak FS High Output platform (Element Biosciences).