title: WGS of Streptococcus pneumoniae BHN97x
design: S. pneumoniae strain BHN97x was grown to an optical density at 600 nm (OD620) of 0.4, corresponding to 108 cells/mL, in C+Y and used to infect three independent lineages of mice. Female BALB/cJ mice (Jackson Laboratory, Bar Harbor, ME) aged seven weeks were maintained in BSL2 facilities. All experiments were done under inhaled isoflurane (2.5%). Mice were challenged intranasally with 5  105 colony forming units (CFUs) of BHN97x in 100 mL phosphate-buffered saline (PBS) as described previously33. The BHN97x challenge strains had been engineered to express luciferase as described34. At 72 h post challenge mice were euthanized and bacteria recovered from the nasal passages via retrotracheal lavage with 3 mLs cold PBS. Nasal lavage was centrifuged for 5 min at 300  g to pellet host cells. Supernatant was transferred and bacterial cells harvested by 15,000xg centrifugation for 5 min. Bacterial pellets were resuspended in 520 mL ThyB and harvested bacterial were plated on triplicate TSA blood agar plates for enumeration of recovery and to expand the population for the subsequent round of infection. Bacterial populations were recovered following overnight incubation at 37 C in 5% CO2 and resuspended in C+Y media. The bacterial suspension was adjusted to OD600 = 0.4 to normalize the following round of infections. Bacterial inoculum and total recovered bacterial populations were enumerated for each infection and subsequent recovery by serial dilution and plating. A total of three experimental evolutionary lineages were maintained for a total of ten generations.