NextSeq 500 sequencing; Identification of adaptive mechanisms leading to reduced antibiotic susceptibility in bacterial biofilms using experimental evolution. Part 1: Pseudomonas aeruginosa strain AA2 biofilms on beads
Identification of adaptive mechanisms leading to reduced antibiotic susceptibility in bacterial biofilms using experimental evolution. Part 1: Pseudomonas aeruginosa strain AA2 biofilms on beads
Evolved bacterial lineages were grown in liquid BHI medium over night, pelleted, and immediately submitted to DNA extraction. Genomic DNA was extracted from 2 mL planktonic cultures. The pellet was resuspended in TE buffer (10 mM Tris-HCl pH 8 + 1 mM EDTA pH 8). 100 µL cell suspension was added to  500 µL lysis buffer (50 mM Tris-HCl pH 8, 70 mM EDTA pH 8, 1% sodium dodecyl sulfate) with 0.5 µg/mL pronase (Roche, Germany) containing acid-washed glass beads (Sigma-Aldrich, USA). After vortexing, the tubes were incubated at 37°C for 1 hour. 200 µL saturated ammonium acetate was added, vortexed, then 600 µL chloroform was added, and vortexed. After centrifuging for 5 minutes 400 µL of the top aqueous phase was transferred to a new tube containing 1 mL 100% ethanol. This was mixed by inversion and centrifuged for 5 minutes. The pellet was washed with 70% ethanol, and air-dried. The DNA was dissolved in low-EDTA-TE buffer (10 mM Tris-HCl pH 8 + 0.1 mM EDTA pH 8 + 0.5 µg/mL) containing RNase A (Qiagen, Germany), incubated at 37°C for 1 hour. NEBNext Ultra II library prep with 460-1000 ng input, according to the protocol with size selection using ampure XP beads after adapter ligation. PCR-free.