NextSeq 500 sequencing; Identification of adaptive mechanisms leading to reduced antibiotic susceptibility in bacterial biofilms using experimental evolution. Part 3: Pseudomonas aeruginosa floating biofilms in synthetic cystic fibrosis medium
Identification of adaptive mechanisms leading to reduced antibiotic susceptibility in bacterial biofilms using experimental evolution. Part 3: Pseudomonas aeruginosa floating biofilms in synthetic cystic fibrosis medium
Evolved bacterial lineages were grown in tryptic soy broth 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.