2D- Day 15
Sequencing was performed on the ancestor clone as well as four evolutionary replicates of each TOB-exposed condition and two replicates of each condition without TOB exposure from day 30. To prepare for sequencing, freezer stocks of populations were revived into liquid culture into the media and condition from which they were evolved and incubated overnight at 37C. The overnight cultures were then sampled by taking an aliquot of culture for planktonic populations or transferring two biofilm beads to 1.0 mL of PBS, sonicating, and using the resuspension for biofilm populations. The Qiagen DNeasy blood and tissue kit was used for DNA extraction. The extracted DNA was then sent to Microbial Genome Sequencing Center for sequencing library preparation and sequencing on an Illumina NextSeq20500.