ERS5792976 SAMEA8105902 208964 Pseudomonas aeruginosa PAO1 Protocols: The biofilm is treated for 24h with tobramycin and C-30 alone or a combination of both for 24h. A sample of the treated biofilm is inoculated in LB and incubated overnight. The DNA-extraction is performed on 3ml of this overnight culture with OD590 = 2. P. aeruginosa PAO1 biofilms were grown in the synthetic cystic fibrosis sputum medium (SCFM2) for 24h. The biofilm was grown in a flat bottom 96-well plate and the inoculum was approx. 5 x 107 CFU/ml. P. aeruginosa biofilms were treated with tobramycin, furanone C-30 or a combination of both in fresh SCFM2 to obtain a final concentration of 20 µg/ml tobramycin and 100 µg/ml furanone C-30. The negative control was treated with fresh SCFM2, including the same amount of DMSO (0.25%) as for the biofilms treated with C-30. After 24 h of static incubation at 37°C, biofilms were sonicated and vortexed in order to disintegrate the biofilm aggregates. A sample from this biofilm was used to start a new cycle. For each treatment three independent lineages were established, that were each exposed for 16 cycles. A sample from the treated biofilm was regrown overnight in LB and 3 ml of a culture with OD590 = 2 was resuspended in 200 µl Tris- EDTA buffer (1mM EDTA, 10 mM Tris-HCl, pH 8.5; Gibco). 100 µl of this culture was added to 500 µl of lysis buffer (50 mM Tris-HCl pH 8, 70 mM EDTA pH 8, 1% sodium dodecyl sulfate, Sigma), containing 0.5 µg/ml pronase (Roche, Mannheim, Germany) and acid washed glass beads (Sigma). After vigorous vortexing for 10 s, the samples were incubated at 37°C during 4 min. Next, 400 µl of saturated ammonium acetate was added and the samples were vortexed for 10 s prior to centrifugation (2 min, 13000 rpm). Subsequently, 600 µl of chloroform was added and the samples were vortexed for another 10 s. The phases were separated by centrifugation (5 min, 13000 rpm) and 400 µl of the top aqueous phase was transferred to a DNAse free Eppendorf tube (Lobind tube, Eppendorf, Aarschot, Belgium) containing 1 ml of 100% ethanol. After centrifugation (5 min, 13000 rpm) the pellet was washed with 500 µl of 70% ethanol and dried in a SpeedVac (Thermo Fisher Scientific, USA) for 10 min. Finally, the extracted DNA was dissolved in 100 µl of low EDTA- Tris buffer (0.1 mM EDTA, 10 mM Tris-HCl, pH 8.5) containing 0.5 µg/ml RNAse (Qiagen, Venlo, The Netherlands) and was incubated at 37°C for 1 h. The DNA was subsequently quantified using a BioDrop µLITE (BioDrop, Cambridge, UK). NEB prep kit ENA first public 2021-03-05 ENA last update 2021-02-18 External Id SAMEA8105902 INSDC center alias Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC center name Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC first public 2021-03-05T04:11:57Z INSDC last update 2021-02-18T14:20:21Z INSDC status public Submitter Id E-MTAB-10124:L1C10 C30 broker name ArrayExpress compound DMSO dose 0.25 genotype wild type genotype growth condition SCFM2 individual 1 organism part whole organism passage 10 sample name E-MTAB-10124:L1C10 C30 stimulus furanone C-30; 100 microgram per mililiter strain PAO1 ERS5792977 SAMEA8105903 208964 Pseudomonas aeruginosa PAO1 Protocols: The biofilm is treated for 24h with tobramycin and C-30 alone or a combination of both for 24h. A sample of the treated biofilm is inoculated in LB and incubated overnight. The DNA-extraction is performed on 3ml of this overnight culture with OD590 = 2. P. aeruginosa PAO1 biofilms were grown in the synthetic cystic fibrosis sputum medium (SCFM2) for 24h. The biofilm was grown in a flat bottom 96-well plate and the inoculum was approx. 5 x 107 CFU/ml. P. aeruginosa biofilms were treated with tobramycin, furanone C-30 or a combination of both in fresh SCFM2 to obtain a final concentration of 20 µg/ml tobramycin and 100 µg/ml furanone C-30. The negative control was treated with fresh SCFM2, including the same amount of DMSO (0.25%) as for the biofilms treated with C-30. After 24 h of static incubation at 37°C, biofilms were sonicated and vortexed in order to disintegrate the biofilm aggregates. A sample from this biofilm was used to start a new cycle. For each treatment three independent lineages were established, that were each exposed for 16 cycles. A sample from the treated biofilm was regrown overnight in LB and 3 ml of a culture with OD590 = 2 was resuspended in 200 µl Tris- EDTA buffer (1mM EDTA, 10 mM Tris-HCl, pH 8.5; Gibco). 100 µl of this culture was added to 500 µl of lysis buffer (50 mM Tris-HCl pH 8, 70 mM EDTA pH 8, 1% sodium dodecyl sulfate, Sigma), containing 0.5 µg/ml pronase (Roche, Mannheim, Germany) and acid washed glass beads (Sigma). After vigorous vortexing for 10 s, the samples were incubated at 37°C during 4 min. Next, 400 µl of saturated ammonium acetate was added and the samples were vortexed for 10 s prior to centrifugation (2 min, 13000 rpm). Subsequently, 600 µl of chloroform was added and the samples were vortexed for another 10 s. The phases were separated by centrifugation (5 min, 13000 rpm) and 400 µl of the top aqueous phase was transferred to a DNAse free Eppendorf tube (Lobind tube, Eppendorf, Aarschot, Belgium) containing 1 ml of 100% ethanol. After centrifugation (5 min, 13000 rpm) the pellet was washed with 500 µl of 70% ethanol and dried in a SpeedVac (Thermo Fisher Scientific, USA) for 10 min. Finally, the extracted DNA was dissolved in 100 µl of low EDTA- Tris buffer (0.1 mM EDTA, 10 mM Tris-HCl, pH 8.5) containing 0.5 µg/ml RNAse (Qiagen, Venlo, The Netherlands) and was incubated at 37°C for 1 h. The DNA was subsequently quantified using a BioDrop µLITE (BioDrop, Cambridge, UK). NEB prep kit ENA first public 2021-03-05 ENA last update 2021-02-18 External Id SAMEA8105903 INSDC center alias Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC center name Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC first public 2021-03-05T04:11:57Z INSDC last update 2021-02-18T14:20:21Z INSDC status public Submitter Id E-MTAB-10124:L1C10 control broker name ArrayExpress compound DMSO dose 0.25 genotype wild type genotype growth condition SCFM2 individual 1 organism part whole organism passage 10 sample name E-MTAB-10124:L1C10 control strain PAO1 ERS5792978 SAMEA8105904 208964 Pseudomonas aeruginosa PAO1 Protocols: The biofilm is treated for 24h with tobramycin and C-30 alone or a combination of both for 24h. A sample of the treated biofilm is inoculated in LB and incubated overnight. The DNA-extraction is performed on 3ml of this overnight culture with OD590 = 2. P. aeruginosa PAO1 biofilms were grown in the synthetic cystic fibrosis sputum medium (SCFM2) for 24h. The biofilm was grown in a flat bottom 96-well plate and the inoculum was approx. 5 x 107 CFU/ml. P. aeruginosa biofilms were treated with tobramycin, furanone C-30 or a combination of both in fresh SCFM2 to obtain a final concentration of 20 µg/ml tobramycin and 100 µg/ml furanone C-30. The negative control was treated with fresh SCFM2, including the same amount of DMSO (0.25%) as for the biofilms treated with C-30. After 24 h of static incubation at 37°C, biofilms were sonicated and vortexed in order to disintegrate the biofilm aggregates. A sample from this biofilm was used to start a new cycle. For each treatment three independent lineages were established, that were each exposed for 16 cycles. A sample from the treated biofilm was regrown overnight in LB and 3 ml of a culture with OD590 = 2 was resuspended in 200 µl Tris- EDTA buffer (1mM EDTA, 10 mM Tris-HCl, pH 8.5; Gibco). 100 µl of this culture was added to 500 µl of lysis buffer (50 mM Tris-HCl pH 8, 70 mM EDTA pH 8, 1% sodium dodecyl sulfate, Sigma), containing 0.5 µg/ml pronase (Roche, Mannheim, Germany) and acid washed glass beads (Sigma). After vigorous vortexing for 10 s, the samples were incubated at 37°C during 4 min. Next, 400 µl of saturated ammonium acetate was added and the samples were vortexed for 10 s prior to centrifugation (2 min, 13000 rpm). Subsequently, 600 µl of chloroform was added and the samples were vortexed for another 10 s. The phases were separated by centrifugation (5 min, 13000 rpm) and 400 µl of the top aqueous phase was transferred to a DNAse free Eppendorf tube (Lobind tube, Eppendorf, Aarschot, Belgium) containing 1 ml of 100% ethanol. After centrifugation (5 min, 13000 rpm) the pellet was washed with 500 µl of 70% ethanol and dried in a SpeedVac (Thermo Fisher Scientific, USA) for 10 min. Finally, the extracted DNA was dissolved in 100 µl of low EDTA- Tris buffer (0.1 mM EDTA, 10 mM Tris-HCl, pH 8.5) containing 0.5 µg/ml RNAse (Qiagen, Venlo, The Netherlands) and was incubated at 37°C for 1 h. The DNA was subsequently quantified using a BioDrop µLITE (BioDrop, Cambridge, UK). NEB prep kit ENA first public 2021-03-05 ENA last update 2021-02-18 External Id SAMEA8105904 INSDC center alias Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC center name Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC first public 2021-03-05T04:11:57Z INSDC last update 2021-02-18T14:20:21Z INSDC status public Submitter Id E-MTAB-10124:L1C10 tob broker name ArrayExpress compound tobramycin dose 20 genotype wild type genotype growth condition SCFM2 individual 1 organism part whole organism passage 10 sample name E-MTAB-10124:L1C10 tob strain PAO1 ERS5792979 SAMEA8105905 208964 Pseudomonas aeruginosa PAO1 Protocols: The biofilm is treated for 24h with tobramycin and C-30 alone or a combination of both for 24h. A sample of the treated biofilm is inoculated in LB and incubated overnight. The DNA-extraction is performed on 3ml of this overnight culture with OD590 = 2. P. aeruginosa PAO1 biofilms were grown in the synthetic cystic fibrosis sputum medium (SCFM2) for 24h. The biofilm was grown in a flat bottom 96-well plate and the inoculum was approx. 5 x 107 CFU/ml. P. aeruginosa biofilms were treated with tobramycin, furanone C-30 or a combination of both in fresh SCFM2 to obtain a final concentration of 20 µg/ml tobramycin and 100 µg/ml furanone C-30. The negative control was treated with fresh SCFM2, including the same amount of DMSO (0.25%) as for the biofilms treated with C-30. After 24 h of static incubation at 37°C, biofilms were sonicated and vortexed in order to disintegrate the biofilm aggregates. A sample from this biofilm was used to start a new cycle. For each treatment three independent lineages were established, that were each exposed for 16 cycles. A sample from the treated biofilm was regrown overnight in LB and 3 ml of a culture with OD590 = 2 was resuspended in 200 µl Tris- EDTA buffer (1mM EDTA, 10 mM Tris-HCl, pH 8.5; Gibco). 100 µl of this culture was added to 500 µl of lysis buffer (50 mM Tris-HCl pH 8, 70 mM EDTA pH 8, 1% sodium dodecyl sulfate, Sigma), containing 0.5 µg/ml pronase (Roche, Mannheim, Germany) and acid washed glass beads (Sigma). After vigorous vortexing for 10 s, the samples were incubated at 37°C during 4 min. Next, 400 µl of saturated ammonium acetate was added and the samples were vortexed for 10 s prior to centrifugation (2 min, 13000 rpm). Subsequently, 600 µl of chloroform was added and the samples were vortexed for another 10 s. The phases were separated by centrifugation (5 min, 13000 rpm) and 400 µl of the top aqueous phase was transferred to a DNAse free Eppendorf tube (Lobind tube, Eppendorf, Aarschot, Belgium) containing 1 ml of 100% ethanol. After centrifugation (5 min, 13000 rpm) the pellet was washed with 500 µl of 70% ethanol and dried in a SpeedVac (Thermo Fisher Scientific, USA) for 10 min. Finally, the extracted DNA was dissolved in 100 µl of low EDTA- Tris buffer (0.1 mM EDTA, 10 mM Tris-HCl, pH 8.5) containing 0.5 µg/ml RNAse (Qiagen, Venlo, The Netherlands) and was incubated at 37°C for 1 h. The DNA was subsequently quantified using a BioDrop µLITE (BioDrop, Cambridge, UK). NEB prep kit ENA first public 2021-03-05 ENA last update 2021-02-18 External Id SAMEA8105905 INSDC center alias Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC center name Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC first public 2021-03-05T04:11:57Z INSDC last update 2021-02-18T14:20:21Z INSDC status public Submitter Id E-MTAB-10124:L1C10 tob + C30 broker name ArrayExpress compound tobramycin; 20 microgram per mililiter and DMSO; 0.25 percent genotype wild type genotype growth condition SCFM2 individual 1 organism part whole organism passage 10 sample name E-MTAB-10124:L1C10 tob + C30 stimulus furanone C-30; 100 microgram per mililiter strain PAO1 ERS5792980 SAMEA8105906 208964 Pseudomonas aeruginosa PAO1 Protocols: The biofilm is treated for 24h with tobramycin and C-30 alone or a combination of both for 24h. A sample of the treated biofilm is inoculated in LB and incubated overnight. The DNA-extraction is performed on 3ml of this overnight culture with OD590 = 2. P. aeruginosa PAO1 biofilms were grown in the synthetic cystic fibrosis sputum medium (SCFM2) for 24h. The biofilm was grown in a flat bottom 96-well plate and the inoculum was approx. 5 x 107 CFU/ml. P. aeruginosa biofilms were treated with tobramycin, furanone C-30 or a combination of both in fresh SCFM2 to obtain a final concentration of 20 µg/ml tobramycin and 100 µg/ml furanone C-30. The negative control was treated with fresh SCFM2, including the same amount of DMSO (0.25%) as for the biofilms treated with C-30. After 24 h of static incubation at 37°C, biofilms were sonicated and vortexed in order to disintegrate the biofilm aggregates. A sample from this biofilm was used to start a new cycle. For each treatment three independent lineages were established, that were each exposed for 16 cycles. A sample from the treated biofilm was regrown overnight in LB and 3 ml of a culture with OD590 = 2 was resuspended in 200 µl Tris- EDTA buffer (1mM EDTA, 10 mM Tris-HCl, pH 8.5; Gibco). 100 µl of this culture was added to 500 µl of lysis buffer (50 mM Tris-HCl pH 8, 70 mM EDTA pH 8, 1% sodium dodecyl sulfate, Sigma), containing 0.5 µg/ml pronase (Roche, Mannheim, Germany) and acid washed glass beads (Sigma). After vigorous vortexing for 10 s, the samples were incubated at 37°C during 4 min. Next, 400 µl of saturated ammonium acetate was added and the samples were vortexed for 10 s prior to centrifugation (2 min, 13000 rpm). Subsequently, 600 µl of chloroform was added and the samples were vortexed for another 10 s. The phases were separated by centrifugation (5 min, 13000 rpm) and 400 µl of the top aqueous phase was transferred to a DNAse free Eppendorf tube (Lobind tube, Eppendorf, Aarschot, Belgium) containing 1 ml of 100% ethanol. After centrifugation (5 min, 13000 rpm) the pellet was washed with 500 µl of 70% ethanol and dried in a SpeedVac (Thermo Fisher Scientific, USA) for 10 min. Finally, the extracted DNA was dissolved in 100 µl of low EDTA- Tris buffer (0.1 mM EDTA, 10 mM Tris-HCl, pH 8.5) containing 0.5 µg/ml RNAse (Qiagen, Venlo, The Netherlands) and was incubated at 37°C for 1 h. The DNA was subsequently quantified using a BioDrop µLITE (BioDrop, Cambridge, UK). NEB prep kit ENA first public 2021-03-05 ENA last update 2021-02-18 External Id SAMEA8105906 INSDC center alias Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC center name Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC first public 2021-03-05T04:11:57Z INSDC last update 2021-02-18T14:20:21Z INSDC status public Submitter Id E-MTAB-10124:L1C16 C30 broker name ArrayExpress compound DMSO dose 0.25 genotype wild type genotype growth condition SCFM2 individual 1 organism part whole organism passage 16 sample name E-MTAB-10124:L1C16 C30 stimulus furanone C-30; 100 microgram per mililiter strain PAO1 ERS5792981 SAMEA8105907 208964 Pseudomonas aeruginosa PAO1 Protocols: The biofilm is treated for 24h with tobramycin and C-30 alone or a combination of both for 24h. A sample of the treated biofilm is inoculated in LB and incubated overnight. The DNA-extraction is performed on 3ml of this overnight culture with OD590 = 2. P. aeruginosa PAO1 biofilms were grown in the synthetic cystic fibrosis sputum medium (SCFM2) for 24h. The biofilm was grown in a flat bottom 96-well plate and the inoculum was approx. 5 x 107 CFU/ml. P. aeruginosa biofilms were treated with tobramycin, furanone C-30 or a combination of both in fresh SCFM2 to obtain a final concentration of 20 µg/ml tobramycin and 100 µg/ml furanone C-30. The negative control was treated with fresh SCFM2, including the same amount of DMSO (0.25%) as for the biofilms treated with C-30. After 24 h of static incubation at 37°C, biofilms were sonicated and vortexed in order to disintegrate the biofilm aggregates. A sample from this biofilm was used to start a new cycle. For each treatment three independent lineages were established, that were each exposed for 16 cycles. A sample from the treated biofilm was regrown overnight in LB and 3 ml of a culture with OD590 = 2 was resuspended in 200 µl Tris- EDTA buffer (1mM EDTA, 10 mM Tris-HCl, pH 8.5; Gibco). 100 µl of this culture was added to 500 µl of lysis buffer (50 mM Tris-HCl pH 8, 70 mM EDTA pH 8, 1% sodium dodecyl sulfate, Sigma), containing 0.5 µg/ml pronase (Roche, Mannheim, Germany) and acid washed glass beads (Sigma). After vigorous vortexing for 10 s, the samples were incubated at 37°C during 4 min. Next, 400 µl of saturated ammonium acetate was added and the samples were vortexed for 10 s prior to centrifugation (2 min, 13000 rpm). Subsequently, 600 µl of chloroform was added and the samples were vortexed for another 10 s. The phases were separated by centrifugation (5 min, 13000 rpm) and 400 µl of the top aqueous phase was transferred to a DNAse free Eppendorf tube (Lobind tube, Eppendorf, Aarschot, Belgium) containing 1 ml of 100% ethanol. After centrifugation (5 min, 13000 rpm) the pellet was washed with 500 µl of 70% ethanol and dried in a SpeedVac (Thermo Fisher Scientific, USA) for 10 min. Finally, the extracted DNA was dissolved in 100 µl of low EDTA- Tris buffer (0.1 mM EDTA, 10 mM Tris-HCl, pH 8.5) containing 0.5 µg/ml RNAse (Qiagen, Venlo, The Netherlands) and was incubated at 37°C for 1 h. The DNA was subsequently quantified using a BioDrop µLITE (BioDrop, Cambridge, UK). NEB prep kit ENA first public 2021-03-05 ENA last update 2021-02-18 External Id SAMEA8105907 INSDC center alias Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC center name Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC first public 2021-03-05T04:11:57Z INSDC last update 2021-02-18T14:20:21Z INSDC status public Submitter Id E-MTAB-10124:L1C16 control broker name ArrayExpress compound DMSO dose 0.25 genotype wild type genotype growth condition SCFM2 individual 1 organism part whole organism passage 16 sample name E-MTAB-10124:L1C16 control strain PAO1 ERS5792982 SAMEA8105908 208964 Pseudomonas aeruginosa PAO1 Protocols: The biofilm is treated for 24h with tobramycin and C-30 alone or a combination of both for 24h. A sample of the treated biofilm is inoculated in LB and incubated overnight. The DNA-extraction is performed on 3ml of this overnight culture with OD590 = 2. P. aeruginosa PAO1 biofilms were grown in the synthetic cystic fibrosis sputum medium (SCFM2) for 24h. The biofilm was grown in a flat bottom 96-well plate and the inoculum was approx. 5 x 107 CFU/ml. P. aeruginosa biofilms were treated with tobramycin, furanone C-30 or a combination of both in fresh SCFM2 to obtain a final concentration of 20 µg/ml tobramycin and 100 µg/ml furanone C-30. The negative control was treated with fresh SCFM2, including the same amount of DMSO (0.25%) as for the biofilms treated with C-30. After 24 h of static incubation at 37°C, biofilms were sonicated and vortexed in order to disintegrate the biofilm aggregates. A sample from this biofilm was used to start a new cycle. For each treatment three independent lineages were established, that were each exposed for 16 cycles. A sample from the treated biofilm was regrown overnight in LB and 3 ml of a culture with OD590 = 2 was resuspended in 200 µl Tris- EDTA buffer (1mM EDTA, 10 mM Tris-HCl, pH 8.5; Gibco). 100 µl of this culture was added to 500 µl of lysis buffer (50 mM Tris-HCl pH 8, 70 mM EDTA pH 8, 1% sodium dodecyl sulfate, Sigma), containing 0.5 µg/ml pronase (Roche, Mannheim, Germany) and acid washed glass beads (Sigma). After vigorous vortexing for 10 s, the samples were incubated at 37°C during 4 min. Next, 400 µl of saturated ammonium acetate was added and the samples were vortexed for 10 s prior to centrifugation (2 min, 13000 rpm). Subsequently, 600 µl of chloroform was added and the samples were vortexed for another 10 s. The phases were separated by centrifugation (5 min, 13000 rpm) and 400 µl of the top aqueous phase was transferred to a DNAse free Eppendorf tube (Lobind tube, Eppendorf, Aarschot, Belgium) containing 1 ml of 100% ethanol. After centrifugation (5 min, 13000 rpm) the pellet was washed with 500 µl of 70% ethanol and dried in a SpeedVac (Thermo Fisher Scientific, USA) for 10 min. Finally, the extracted DNA was dissolved in 100 µl of low EDTA- Tris buffer (0.1 mM EDTA, 10 mM Tris-HCl, pH 8.5) containing 0.5 µg/ml RNAse (Qiagen, Venlo, The Netherlands) and was incubated at 37°C for 1 h. The DNA was subsequently quantified using a BioDrop µLITE (BioDrop, Cambridge, UK). NEB prep kit ENA first public 2021-03-05 ENA last update 2021-02-18 External Id SAMEA8105908 INSDC center alias Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC center name Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC first public 2021-03-05T04:11:57Z INSDC last update 2021-02-18T14:20:21Z INSDC status public Submitter Id E-MTAB-10124:L1C16 tob broker name ArrayExpress compound tobramycin dose 20 genotype wild type genotype growth condition SCFM2 individual 1 organism part whole organism passage 16 sample name E-MTAB-10124:L1C16 tob strain PAO1 ERS5792983 SAMEA8105909 208964 Pseudomonas aeruginosa PAO1 Protocols: The biofilm is treated for 24h with tobramycin and C-30 alone or a combination of both for 24h. A sample of the treated biofilm is inoculated in LB and incubated overnight. The DNA-extraction is performed on 3ml of this overnight culture with OD590 = 2. P. aeruginosa PAO1 biofilms were grown in the synthetic cystic fibrosis sputum medium (SCFM2) for 24h. The biofilm was grown in a flat bottom 96-well plate and the inoculum was approx. 5 x 107 CFU/ml. P. aeruginosa biofilms were treated with tobramycin, furanone C-30 or a combination of both in fresh SCFM2 to obtain a final concentration of 20 µg/ml tobramycin and 100 µg/ml furanone C-30. The negative control was treated with fresh SCFM2, including the same amount of DMSO (0.25%) as for the biofilms treated with C-30. After 24 h of static incubation at 37°C, biofilms were sonicated and vortexed in order to disintegrate the biofilm aggregates. A sample from this biofilm was used to start a new cycle. For each treatment three independent lineages were established, that were each exposed for 16 cycles. A sample from the treated biofilm was regrown overnight in LB and 3 ml of a culture with OD590 = 2 was resuspended in 200 µl Tris- EDTA buffer (1mM EDTA, 10 mM Tris-HCl, pH 8.5; Gibco). 100 µl of this culture was added to 500 µl of lysis buffer (50 mM Tris-HCl pH 8, 70 mM EDTA pH 8, 1% sodium dodecyl sulfate, Sigma), containing 0.5 µg/ml pronase (Roche, Mannheim, Germany) and acid washed glass beads (Sigma). After vigorous vortexing for 10 s, the samples were incubated at 37°C during 4 min. Next, 400 µl of saturated ammonium acetate was added and the samples were vortexed for 10 s prior to centrifugation (2 min, 13000 rpm). Subsequently, 600 µl of chloroform was added and the samples were vortexed for another 10 s. The phases were separated by centrifugation (5 min, 13000 rpm) and 400 µl of the top aqueous phase was transferred to a DNAse free Eppendorf tube (Lobind tube, Eppendorf, Aarschot, Belgium) containing 1 ml of 100% ethanol. After centrifugation (5 min, 13000 rpm) the pellet was washed with 500 µl of 70% ethanol and dried in a SpeedVac (Thermo Fisher Scientific, USA) for 10 min. Finally, the extracted DNA was dissolved in 100 µl of low EDTA- Tris buffer (0.1 mM EDTA, 10 mM Tris-HCl, pH 8.5) containing 0.5 µg/ml RNAse (Qiagen, Venlo, The Netherlands) and was incubated at 37°C for 1 h. The DNA was subsequently quantified using a BioDrop µLITE (BioDrop, Cambridge, UK). NEB prep kit ENA first public 2021-03-05 ENA last update 2021-02-18 External Id SAMEA8105909 INSDC center alias Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC center name Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC first public 2021-03-05T04:11:57Z INSDC last update 2021-02-18T14:20:21Z INSDC status public Submitter Id E-MTAB-10124:L1C16 tob + C30 broker name ArrayExpress compound tobramycin; 20 microgram per mililiter and DMSO; 0.25 percent genotype wild type genotype growth condition SCFM2 individual 1 organism part whole organism passage 16 sample name E-MTAB-10124:L1C16 tob + C30 stimulus furanone C-30; 100 microgram per mililiter strain PAO1 ERS5792984 SAMEA8105910 208964 Pseudomonas aeruginosa PAO1 Protocols: The biofilm is treated for 24h with tobramycin and C-30 alone or a combination of both for 24h. A sample of the treated biofilm is inoculated in LB and incubated overnight. The DNA-extraction is performed on 3ml of this overnight culture with OD590 = 2. P. aeruginosa PAO1 biofilms were grown in the synthetic cystic fibrosis sputum medium (SCFM2) for 24h. The biofilm was grown in a flat bottom 96-well plate and the inoculum was approx. 5 x 107 CFU/ml. P. aeruginosa biofilms were treated with tobramycin, furanone C-30 or a combination of both in fresh SCFM2 to obtain a final concentration of 20 µg/ml tobramycin and 100 µg/ml furanone C-30. The negative control was treated with fresh SCFM2, including the same amount of DMSO (0.25%) as for the biofilms treated with C-30. After 24 h of static incubation at 37°C, biofilms were sonicated and vortexed in order to disintegrate the biofilm aggregates. A sample from this biofilm was used to start a new cycle. For each treatment three independent lineages were established, that were each exposed for 16 cycles. A sample from the treated biofilm was regrown overnight in LB and 3 ml of a culture with OD590 = 2 was resuspended in 200 µl Tris- EDTA buffer (1mM EDTA, 10 mM Tris-HCl, pH 8.5; Gibco). 100 µl of this culture was added to 500 µl of lysis buffer (50 mM Tris-HCl pH 8, 70 mM EDTA pH 8, 1% sodium dodecyl sulfate, Sigma), containing 0.5 µg/ml pronase (Roche, Mannheim, Germany) and acid washed glass beads (Sigma). After vigorous vortexing for 10 s, the samples were incubated at 37°C during 4 min. Next, 400 µl of saturated ammonium acetate was added and the samples were vortexed for 10 s prior to centrifugation (2 min, 13000 rpm). Subsequently, 600 µl of chloroform was added and the samples were vortexed for another 10 s. The phases were separated by centrifugation (5 min, 13000 rpm) and 400 µl of the top aqueous phase was transferred to a DNAse free Eppendorf tube (Lobind tube, Eppendorf, Aarschot, Belgium) containing 1 ml of 100% ethanol. After centrifugation (5 min, 13000 rpm) the pellet was washed with 500 µl of 70% ethanol and dried in a SpeedVac (Thermo Fisher Scientific, USA) for 10 min. Finally, the extracted DNA was dissolved in 100 µl of low EDTA- Tris buffer (0.1 mM EDTA, 10 mM Tris-HCl, pH 8.5) containing 0.5 µg/ml RNAse (Qiagen, Venlo, The Netherlands) and was incubated at 37°C for 1 h. The DNA was subsequently quantified using a BioDrop µLITE (BioDrop, Cambridge, UK). NEB prep kit ENA first public 2021-03-05 ENA last update 2021-02-18 External Id SAMEA8105910 INSDC center alias Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC center name Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC first public 2021-03-05T04:11:57Z INSDC last update 2021-02-18T14:20:21Z INSDC status public Submitter Id E-MTAB-10124:L1C5 C30 broker name ArrayExpress compound DMSO dose 0.25 genotype wild type genotype growth condition SCFM2 individual 1 organism part whole organism passage 5 sample name E-MTAB-10124:L1C5 C30 stimulus furanone C-30; 100 microgram per mililiter strain PAO1 ERS5792985 SAMEA8105911 208964 Pseudomonas aeruginosa PAO1 Protocols: The biofilm is treated for 24h with tobramycin and C-30 alone or a combination of both for 24h. A sample of the treated biofilm is inoculated in LB and incubated overnight. The DNA-extraction is performed on 3ml of this overnight culture with OD590 = 2. P. aeruginosa PAO1 biofilms were grown in the synthetic cystic fibrosis sputum medium (SCFM2) for 24h. The biofilm was grown in a flat bottom 96-well plate and the inoculum was approx. 5 x 107 CFU/ml. P. aeruginosa biofilms were treated with tobramycin, furanone C-30 or a combination of both in fresh SCFM2 to obtain a final concentration of 20 µg/ml tobramycin and 100 µg/ml furanone C-30. The negative control was treated with fresh SCFM2, including the same amount of DMSO (0.25%) as for the biofilms treated with C-30. After 24 h of static incubation at 37°C, biofilms were sonicated and vortexed in order to disintegrate the biofilm aggregates. A sample from this biofilm was used to start a new cycle. For each treatment three independent lineages were established, that were each exposed for 16 cycles. A sample from the treated biofilm was regrown overnight in LB and 3 ml of a culture with OD590 = 2 was resuspended in 200 µl Tris- EDTA buffer (1mM EDTA, 10 mM Tris-HCl, pH 8.5; Gibco). 100 µl of this culture was added to 500 µl of lysis buffer (50 mM Tris-HCl pH 8, 70 mM EDTA pH 8, 1% sodium dodecyl sulfate, Sigma), containing 0.5 µg/ml pronase (Roche, Mannheim, Germany) and acid washed glass beads (Sigma). After vigorous vortexing for 10 s, the samples were incubated at 37°C during 4 min. Next, 400 µl of saturated ammonium acetate was added and the samples were vortexed for 10 s prior to centrifugation (2 min, 13000 rpm). Subsequently, 600 µl of chloroform was added and the samples were vortexed for another 10 s. The phases were separated by centrifugation (5 min, 13000 rpm) and 400 µl of the top aqueous phase was transferred to a DNAse free Eppendorf tube (Lobind tube, Eppendorf, Aarschot, Belgium) containing 1 ml of 100% ethanol. After centrifugation (5 min, 13000 rpm) the pellet was washed with 500 µl of 70% ethanol and dried in a SpeedVac (Thermo Fisher Scientific, USA) for 10 min. Finally, the extracted DNA was dissolved in 100 µl of low EDTA- Tris buffer (0.1 mM EDTA, 10 mM Tris-HCl, pH 8.5) containing 0.5 µg/ml RNAse (Qiagen, Venlo, The Netherlands) and was incubated at 37°C for 1 h. The DNA was subsequently quantified using a BioDrop µLITE (BioDrop, Cambridge, UK). NEB prep kit ENA first public 2021-03-05 ENA last update 2021-02-18 External Id SAMEA8105911 INSDC center alias Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC center name Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC first public 2021-03-05T04:11:57Z INSDC last update 2021-02-18T14:20:21Z INSDC status public Submitter Id E-MTAB-10124:L1C5 control broker name ArrayExpress compound DMSO dose 0.25 genotype wild type genotype growth condition SCFM2 individual 1 organism part whole organism passage 5 sample name E-MTAB-10124:L1C5 control strain PAO1 ERS5792986 SAMEA8105912 208964 Pseudomonas aeruginosa PAO1 Protocols: The biofilm is treated for 24h with tobramycin and C-30 alone or a combination of both for 24h. A sample of the treated biofilm is inoculated in LB and incubated overnight. The DNA-extraction is performed on 3ml of this overnight culture with OD590 = 2. P. aeruginosa PAO1 biofilms were grown in the synthetic cystic fibrosis sputum medium (SCFM2) for 24h. The biofilm was grown in a flat bottom 96-well plate and the inoculum was approx. 5 x 107 CFU/ml. P. aeruginosa biofilms were treated with tobramycin, furanone C-30 or a combination of both in fresh SCFM2 to obtain a final concentration of 20 µg/ml tobramycin and 100 µg/ml furanone C-30. The negative control was treated with fresh SCFM2, including the same amount of DMSO (0.25%) as for the biofilms treated with C-30. After 24 h of static incubation at 37°C, biofilms were sonicated and vortexed in order to disintegrate the biofilm aggregates. A sample from this biofilm was used to start a new cycle. For each treatment three independent lineages were established, that were each exposed for 16 cycles. A sample from the treated biofilm was regrown overnight in LB and 3 ml of a culture with OD590 = 2 was resuspended in 200 µl Tris- EDTA buffer (1mM EDTA, 10 mM Tris-HCl, pH 8.5; Gibco). 100 µl of this culture was added to 500 µl of lysis buffer (50 mM Tris-HCl pH 8, 70 mM EDTA pH 8, 1% sodium dodecyl sulfate, Sigma), containing 0.5 µg/ml pronase (Roche, Mannheim, Germany) and acid washed glass beads (Sigma). After vigorous vortexing for 10 s, the samples were incubated at 37°C during 4 min. Next, 400 µl of saturated ammonium acetate was added and the samples were vortexed for 10 s prior to centrifugation (2 min, 13000 rpm). Subsequently, 600 µl of chloroform was added and the samples were vortexed for another 10 s. The phases were separated by centrifugation (5 min, 13000 rpm) and 400 µl of the top aqueous phase was transferred to a DNAse free Eppendorf tube (Lobind tube, Eppendorf, Aarschot, Belgium) containing 1 ml of 100% ethanol. After centrifugation (5 min, 13000 rpm) the pellet was washed with 500 µl of 70% ethanol and dried in a SpeedVac (Thermo Fisher Scientific, USA) for 10 min. Finally, the extracted DNA was dissolved in 100 µl of low EDTA- Tris buffer (0.1 mM EDTA, 10 mM Tris-HCl, pH 8.5) containing 0.5 µg/ml RNAse (Qiagen, Venlo, The Netherlands) and was incubated at 37°C for 1 h. The DNA was subsequently quantified using a BioDrop µLITE (BioDrop, Cambridge, UK). NEB prep kit ENA first public 2021-03-05 ENA last update 2021-02-18 External Id SAMEA8105912 INSDC center alias Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC center name Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC first public 2021-03-05T04:11:57Z INSDC last update 2021-02-18T14:20:21Z INSDC status public Submitter Id E-MTAB-10124:L1C5 tob broker name ArrayExpress compound tobramycin dose 20 genotype wild type genotype growth condition SCFM2 individual 1 organism part whole organism passage 5 sample name E-MTAB-10124:L1C5 tob strain PAO1 ERS5792987 SAMEA8105913 208964 Pseudomonas aeruginosa PAO1 Protocols: The biofilm is treated for 24h with tobramycin and C-30 alone or a combination of both for 24h. A sample of the treated biofilm is inoculated in LB and incubated overnight. The DNA-extraction is performed on 3ml of this overnight culture with OD590 = 2. P. aeruginosa PAO1 biofilms were grown in the synthetic cystic fibrosis sputum medium (SCFM2) for 24h. The biofilm was grown in a flat bottom 96-well plate and the inoculum was approx. 5 x 107 CFU/ml. P. aeruginosa biofilms were treated with tobramycin, furanone C-30 or a combination of both in fresh SCFM2 to obtain a final concentration of 20 µg/ml tobramycin and 100 µg/ml furanone C-30. The negative control was treated with fresh SCFM2, including the same amount of DMSO (0.25%) as for the biofilms treated with C-30. After 24 h of static incubation at 37°C, biofilms were sonicated and vortexed in order to disintegrate the biofilm aggregates. A sample from this biofilm was used to start a new cycle. For each treatment three independent lineages were established, that were each exposed for 16 cycles. A sample from the treated biofilm was regrown overnight in LB and 3 ml of a culture with OD590 = 2 was resuspended in 200 µl Tris- EDTA buffer (1mM EDTA, 10 mM Tris-HCl, pH 8.5; Gibco). 100 µl of this culture was added to 500 µl of lysis buffer (50 mM Tris-HCl pH 8, 70 mM EDTA pH 8, 1% sodium dodecyl sulfate, Sigma), containing 0.5 µg/ml pronase (Roche, Mannheim, Germany) and acid washed glass beads (Sigma). After vigorous vortexing for 10 s, the samples were incubated at 37°C during 4 min. Next, 400 µl of saturated ammonium acetate was added and the samples were vortexed for 10 s prior to centrifugation (2 min, 13000 rpm). Subsequently, 600 µl of chloroform was added and the samples were vortexed for another 10 s. The phases were separated by centrifugation (5 min, 13000 rpm) and 400 µl of the top aqueous phase was transferred to a DNAse free Eppendorf tube (Lobind tube, Eppendorf, Aarschot, Belgium) containing 1 ml of 100% ethanol. After centrifugation (5 min, 13000 rpm) the pellet was washed with 500 µl of 70% ethanol and dried in a SpeedVac (Thermo Fisher Scientific, USA) for 10 min. Finally, the extracted DNA was dissolved in 100 µl of low EDTA- Tris buffer (0.1 mM EDTA, 10 mM Tris-HCl, pH 8.5) containing 0.5 µg/ml RNAse (Qiagen, Venlo, The Netherlands) and was incubated at 37°C for 1 h. The DNA was subsequently quantified using a BioDrop µLITE (BioDrop, Cambridge, UK). NEB prep kit ENA first public 2021-03-05 ENA last update 2021-02-18 External Id SAMEA8105913 INSDC center alias Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC center name Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC first public 2021-03-05T04:11:57Z INSDC last update 2021-02-18T14:20:21Z INSDC status public Submitter Id E-MTAB-10124:L1C5 tob + C30 broker name ArrayExpress compound tobramycin; 20 microgram per mililiter and DMSO; 0.25 percent genotype wild type genotype growth condition SCFM2 individual 1 organism part whole organism passage 5 sample name E-MTAB-10124:L1C5 tob + C30 stimulus furanone C-30; 100 microgram per mililiter strain PAO1 ERS5792988 SAMEA8105914 208964 Pseudomonas aeruginosa PAO1 Protocols: The biofilm is treated for 24h with tobramycin and C-30 alone or a combination of both for 24h. A sample of the treated biofilm is inoculated in LB and incubated overnight. The DNA-extraction is performed on 3ml of this overnight culture with OD590 = 2. P. aeruginosa PAO1 biofilms were grown in the synthetic cystic fibrosis sputum medium (SCFM2) for 24h. The biofilm was grown in a flat bottom 96-well plate and the inoculum was approx. 5 x 107 CFU/ml. P. aeruginosa biofilms were treated with tobramycin, furanone C-30 or a combination of both in fresh SCFM2 to obtain a final concentration of 20 µg/ml tobramycin and 100 µg/ml furanone C-30. The negative control was treated with fresh SCFM2, including the same amount of DMSO (0.25%) as for the biofilms treated with C-30. After 24 h of static incubation at 37°C, biofilms were sonicated and vortexed in order to disintegrate the biofilm aggregates. A sample from this biofilm was used to start a new cycle. For each treatment three independent lineages were established, that were each exposed for 16 cycles. A sample from the treated biofilm was regrown overnight in LB and 3 ml of a culture with OD590 = 2 was resuspended in 200 µl Tris- EDTA buffer (1mM EDTA, 10 mM Tris-HCl, pH 8.5; Gibco). 100 µl of this culture was added to 500 µl of lysis buffer (50 mM Tris-HCl pH 8, 70 mM EDTA pH 8, 1% sodium dodecyl sulfate, Sigma), containing 0.5 µg/ml pronase (Roche, Mannheim, Germany) and acid washed glass beads (Sigma). After vigorous vortexing for 10 s, the samples were incubated at 37°C during 4 min. Next, 400 µl of saturated ammonium acetate was added and the samples were vortexed for 10 s prior to centrifugation (2 min, 13000 rpm). Subsequently, 600 µl of chloroform was added and the samples were vortexed for another 10 s. The phases were separated by centrifugation (5 min, 13000 rpm) and 400 µl of the top aqueous phase was transferred to a DNAse free Eppendorf tube (Lobind tube, Eppendorf, Aarschot, Belgium) containing 1 ml of 100% ethanol. After centrifugation (5 min, 13000 rpm) the pellet was washed with 500 µl of 70% ethanol and dried in a SpeedVac (Thermo Fisher Scientific, USA) for 10 min. Finally, the extracted DNA was dissolved in 100 µl of low EDTA- Tris buffer (0.1 mM EDTA, 10 mM Tris-HCl, pH 8.5) containing 0.5 µg/ml RNAse (Qiagen, Venlo, The Netherlands) and was incubated at 37°C for 1 h. The DNA was subsequently quantified using a BioDrop µLITE (BioDrop, Cambridge, UK). NEB prep kit ENA first public 2021-03-05 ENA last update 2021-02-18 External Id SAMEA8105914 INSDC center alias Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC center name Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC first public 2021-03-05T04:11:57Z INSDC last update 2021-02-18T14:20:21Z INSDC status public Submitter Id E-MTAB-10124:L2C10 C30 broker name ArrayExpress compound DMSO dose 0.25 genotype wild type genotype growth condition SCFM2 individual 2 organism part whole organism passage 10 sample name E-MTAB-10124:L2C10 C30 stimulus furanone C-30; 100 microgram per mililiter strain PAO1 ERS5792989 SAMEA8105915 208964 Pseudomonas aeruginosa PAO1 Protocols: The biofilm is treated for 24h with tobramycin and C-30 alone or a combination of both for 24h. A sample of the treated biofilm is inoculated in LB and incubated overnight. The DNA-extraction is performed on 3ml of this overnight culture with OD590 = 2. P. aeruginosa PAO1 biofilms were grown in the synthetic cystic fibrosis sputum medium (SCFM2) for 24h. The biofilm was grown in a flat bottom 96-well plate and the inoculum was approx. 5 x 107 CFU/ml. P. aeruginosa biofilms were treated with tobramycin, furanone C-30 or a combination of both in fresh SCFM2 to obtain a final concentration of 20 µg/ml tobramycin and 100 µg/ml furanone C-30. The negative control was treated with fresh SCFM2, including the same amount of DMSO (0.25%) as for the biofilms treated with C-30. After 24 h of static incubation at 37°C, biofilms were sonicated and vortexed in order to disintegrate the biofilm aggregates. A sample from this biofilm was used to start a new cycle. For each treatment three independent lineages were established, that were each exposed for 16 cycles. A sample from the treated biofilm was regrown overnight in LB and 3 ml of a culture with OD590 = 2 was resuspended in 200 µl Tris- EDTA buffer (1mM EDTA, 10 mM Tris-HCl, pH 8.5; Gibco). 100 µl of this culture was added to 500 µl of lysis buffer (50 mM Tris-HCl pH 8, 70 mM EDTA pH 8, 1% sodium dodecyl sulfate, Sigma), containing 0.5 µg/ml pronase (Roche, Mannheim, Germany) and acid washed glass beads (Sigma). After vigorous vortexing for 10 s, the samples were incubated at 37°C during 4 min. Next, 400 µl of saturated ammonium acetate was added and the samples were vortexed for 10 s prior to centrifugation (2 min, 13000 rpm). Subsequently, 600 µl of chloroform was added and the samples were vortexed for another 10 s. The phases were separated by centrifugation (5 min, 13000 rpm) and 400 µl of the top aqueous phase was transferred to a DNAse free Eppendorf tube (Lobind tube, Eppendorf, Aarschot, Belgium) containing 1 ml of 100% ethanol. After centrifugation (5 min, 13000 rpm) the pellet was washed with 500 µl of 70% ethanol and dried in a SpeedVac (Thermo Fisher Scientific, USA) for 10 min. Finally, the extracted DNA was dissolved in 100 µl of low EDTA- Tris buffer (0.1 mM EDTA, 10 mM Tris-HCl, pH 8.5) containing 0.5 µg/ml RNAse (Qiagen, Venlo, The Netherlands) and was incubated at 37°C for 1 h. The DNA was subsequently quantified using a BioDrop µLITE (BioDrop, Cambridge, UK). NEB prep kit ENA first public 2021-03-05 ENA last update 2021-02-18 External Id SAMEA8105915 INSDC center alias Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC center name Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC first public 2021-03-05T04:11:57Z INSDC last update 2021-02-18T14:20:21Z INSDC status public Submitter Id E-MTAB-10124:L2C10 control broker name ArrayExpress compound DMSO dose 0.25 genotype wild type genotype growth condition SCFM2 individual 2 organism part whole organism passage 10 sample name E-MTAB-10124:L2C10 control strain PAO1 ERS5792990 SAMEA8105916 208964 Pseudomonas aeruginosa PAO1 Protocols: The biofilm is treated for 24h with tobramycin and C-30 alone or a combination of both for 24h. A sample of the treated biofilm is inoculated in LB and incubated overnight. The DNA-extraction is performed on 3ml of this overnight culture with OD590 = 2. P. aeruginosa PAO1 biofilms were grown in the synthetic cystic fibrosis sputum medium (SCFM2) for 24h. The biofilm was grown in a flat bottom 96-well plate and the inoculum was approx. 5 x 107 CFU/ml. P. aeruginosa biofilms were treated with tobramycin, furanone C-30 or a combination of both in fresh SCFM2 to obtain a final concentration of 20 µg/ml tobramycin and 100 µg/ml furanone C-30. The negative control was treated with fresh SCFM2, including the same amount of DMSO (0.25%) as for the biofilms treated with C-30. After 24 h of static incubation at 37°C, biofilms were sonicated and vortexed in order to disintegrate the biofilm aggregates. A sample from this biofilm was used to start a new cycle. For each treatment three independent lineages were established, that were each exposed for 16 cycles. A sample from the treated biofilm was regrown overnight in LB and 3 ml of a culture with OD590 = 2 was resuspended in 200 µl Tris- EDTA buffer (1mM EDTA, 10 mM Tris-HCl, pH 8.5; Gibco). 100 µl of this culture was added to 500 µl of lysis buffer (50 mM Tris-HCl pH 8, 70 mM EDTA pH 8, 1% sodium dodecyl sulfate, Sigma), containing 0.5 µg/ml pronase (Roche, Mannheim, Germany) and acid washed glass beads (Sigma). After vigorous vortexing for 10 s, the samples were incubated at 37°C during 4 min. Next, 400 µl of saturated ammonium acetate was added and the samples were vortexed for 10 s prior to centrifugation (2 min, 13000 rpm). Subsequently, 600 µl of chloroform was added and the samples were vortexed for another 10 s. The phases were separated by centrifugation (5 min, 13000 rpm) and 400 µl of the top aqueous phase was transferred to a DNAse free Eppendorf tube (Lobind tube, Eppendorf, Aarschot, Belgium) containing 1 ml of 100% ethanol. After centrifugation (5 min, 13000 rpm) the pellet was washed with 500 µl of 70% ethanol and dried in a SpeedVac (Thermo Fisher Scientific, USA) for 10 min. Finally, the extracted DNA was dissolved in 100 µl of low EDTA- Tris buffer (0.1 mM EDTA, 10 mM Tris-HCl, pH 8.5) containing 0.5 µg/ml RNAse (Qiagen, Venlo, The Netherlands) and was incubated at 37°C for 1 h. The DNA was subsequently quantified using a BioDrop µLITE (BioDrop, Cambridge, UK). NEB prep kit ENA first public 2021-03-05 ENA last update 2021-02-18 External Id SAMEA8105916 INSDC center alias Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC center name Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC first public 2021-03-05T04:11:57Z INSDC last update 2021-02-18T14:20:21Z INSDC status public Submitter Id E-MTAB-10124:L2C10 tob broker name ArrayExpress compound tobramycin dose 20 genotype wild type genotype growth condition SCFM2 individual 2 organism part whole organism passage 10 sample name E-MTAB-10124:L2C10 tob strain PAO1 ERS5792991 SAMEA8105917 208964 Pseudomonas aeruginosa PAO1 Protocols: The biofilm is treated for 24h with tobramycin and C-30 alone or a combination of both for 24h. A sample of the treated biofilm is inoculated in LB and incubated overnight. The DNA-extraction is performed on 3ml of this overnight culture with OD590 = 2. P. aeruginosa PAO1 biofilms were grown in the synthetic cystic fibrosis sputum medium (SCFM2) for 24h. The biofilm was grown in a flat bottom 96-well plate and the inoculum was approx. 5 x 107 CFU/ml. P. aeruginosa biofilms were treated with tobramycin, furanone C-30 or a combination of both in fresh SCFM2 to obtain a final concentration of 20 µg/ml tobramycin and 100 µg/ml furanone C-30. The negative control was treated with fresh SCFM2, including the same amount of DMSO (0.25%) as for the biofilms treated with C-30. After 24 h of static incubation at 37°C, biofilms were sonicated and vortexed in order to disintegrate the biofilm aggregates. A sample from this biofilm was used to start a new cycle. For each treatment three independent lineages were established, that were each exposed for 16 cycles. A sample from the treated biofilm was regrown overnight in LB and 3 ml of a culture with OD590 = 2 was resuspended in 200 µl Tris- EDTA buffer (1mM EDTA, 10 mM Tris-HCl, pH 8.5; Gibco). 100 µl of this culture was added to 500 µl of lysis buffer (50 mM Tris-HCl pH 8, 70 mM EDTA pH 8, 1% sodium dodecyl sulfate, Sigma), containing 0.5 µg/ml pronase (Roche, Mannheim, Germany) and acid washed glass beads (Sigma). After vigorous vortexing for 10 s, the samples were incubated at 37°C during 4 min. Next, 400 µl of saturated ammonium acetate was added and the samples were vortexed for 10 s prior to centrifugation (2 min, 13000 rpm). Subsequently, 600 µl of chloroform was added and the samples were vortexed for another 10 s. The phases were separated by centrifugation (5 min, 13000 rpm) and 400 µl of the top aqueous phase was transferred to a DNAse free Eppendorf tube (Lobind tube, Eppendorf, Aarschot, Belgium) containing 1 ml of 100% ethanol. After centrifugation (5 min, 13000 rpm) the pellet was washed with 500 µl of 70% ethanol and dried in a SpeedVac (Thermo Fisher Scientific, USA) for 10 min. Finally, the extracted DNA was dissolved in 100 µl of low EDTA- Tris buffer (0.1 mM EDTA, 10 mM Tris-HCl, pH 8.5) containing 0.5 µg/ml RNAse (Qiagen, Venlo, The Netherlands) and was incubated at 37°C for 1 h. The DNA was subsequently quantified using a BioDrop µLITE (BioDrop, Cambridge, UK). NEB prep kit ENA first public 2021-03-05 ENA last update 2021-02-18 External Id SAMEA8105917 INSDC center alias Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC center name Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC first public 2021-03-05T04:11:57Z INSDC last update 2021-02-18T14:20:21Z INSDC status public Submitter Id E-MTAB-10124:L2C10 tob + C30 broker name ArrayExpress compound tobramycin; 20 microgram per mililiter and DMSO; 0.25 percent genotype wild type genotype growth condition SCFM2 individual 2 organism part whole organism passage 10 sample name E-MTAB-10124:L2C10 tob + C30 stimulus furanone C-30; 100 microgram per mililiter strain PAO1 ERS5792992 SAMEA8105918 208964 Pseudomonas aeruginosa PAO1 Protocols: The biofilm is treated for 24h with tobramycin and C-30 alone or a combination of both for 24h. A sample of the treated biofilm is inoculated in LB and incubated overnight. The DNA-extraction is performed on 3ml of this overnight culture with OD590 = 2. P. aeruginosa PAO1 biofilms were grown in the synthetic cystic fibrosis sputum medium (SCFM2) for 24h. The biofilm was grown in a flat bottom 96-well plate and the inoculum was approx. 5 x 107 CFU/ml. P. aeruginosa biofilms were treated with tobramycin, furanone C-30 or a combination of both in fresh SCFM2 to obtain a final concentration of 20 µg/ml tobramycin and 100 µg/ml furanone C-30. The negative control was treated with fresh SCFM2, including the same amount of DMSO (0.25%) as for the biofilms treated with C-30. After 24 h of static incubation at 37°C, biofilms were sonicated and vortexed in order to disintegrate the biofilm aggregates. A sample from this biofilm was used to start a new cycle. For each treatment three independent lineages were established, that were each exposed for 16 cycles. A sample from the treated biofilm was regrown overnight in LB and 3 ml of a culture with OD590 = 2 was resuspended in 200 µl Tris- EDTA buffer (1mM EDTA, 10 mM Tris-HCl, pH 8.5; Gibco). 100 µl of this culture was added to 500 µl of lysis buffer (50 mM Tris-HCl pH 8, 70 mM EDTA pH 8, 1% sodium dodecyl sulfate, Sigma), containing 0.5 µg/ml pronase (Roche, Mannheim, Germany) and acid washed glass beads (Sigma). After vigorous vortexing for 10 s, the samples were incubated at 37°C during 4 min. Next, 400 µl of saturated ammonium acetate was added and the samples were vortexed for 10 s prior to centrifugation (2 min, 13000 rpm). Subsequently, 600 µl of chloroform was added and the samples were vortexed for another 10 s. The phases were separated by centrifugation (5 min, 13000 rpm) and 400 µl of the top aqueous phase was transferred to a DNAse free Eppendorf tube (Lobind tube, Eppendorf, Aarschot, Belgium) containing 1 ml of 100% ethanol. After centrifugation (5 min, 13000 rpm) the pellet was washed with 500 µl of 70% ethanol and dried in a SpeedVac (Thermo Fisher Scientific, USA) for 10 min. Finally, the extracted DNA was dissolved in 100 µl of low EDTA- Tris buffer (0.1 mM EDTA, 10 mM Tris-HCl, pH 8.5) containing 0.5 µg/ml RNAse (Qiagen, Venlo, The Netherlands) and was incubated at 37°C for 1 h. The DNA was subsequently quantified using a BioDrop µLITE (BioDrop, Cambridge, UK). NEB prep kit ENA first public 2021-03-05 ENA last update 2021-02-18 External Id SAMEA8105918 INSDC center alias Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC center name Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC first public 2021-03-05T04:11:57Z INSDC last update 2021-02-18T14:20:21Z INSDC status public Submitter Id E-MTAB-10124:L2C16 C30 broker name ArrayExpress compound DMSO dose 0.25 genotype wild type genotype growth condition SCFM2 individual 2 organism part whole organism passage 16 sample name E-MTAB-10124:L2C16 C30 stimulus furanone C-30; 100 microgram per mililiter strain PAO1 ERS5792993 SAMEA8105919 208964 Pseudomonas aeruginosa PAO1 Protocols: The biofilm is treated for 24h with tobramycin and C-30 alone or a combination of both for 24h. A sample of the treated biofilm is inoculated in LB and incubated overnight. The DNA-extraction is performed on 3ml of this overnight culture with OD590 = 2. P. aeruginosa PAO1 biofilms were grown in the synthetic cystic fibrosis sputum medium (SCFM2) for 24h. The biofilm was grown in a flat bottom 96-well plate and the inoculum was approx. 5 x 107 CFU/ml. P. aeruginosa biofilms were treated with tobramycin, furanone C-30 or a combination of both in fresh SCFM2 to obtain a final concentration of 20 µg/ml tobramycin and 100 µg/ml furanone C-30. The negative control was treated with fresh SCFM2, including the same amount of DMSO (0.25%) as for the biofilms treated with C-30. After 24 h of static incubation at 37°C, biofilms were sonicated and vortexed in order to disintegrate the biofilm aggregates. A sample from this biofilm was used to start a new cycle. For each treatment three independent lineages were established, that were each exposed for 16 cycles. A sample from the treated biofilm was regrown overnight in LB and 3 ml of a culture with OD590 = 2 was resuspended in 200 µl Tris- EDTA buffer (1mM EDTA, 10 mM Tris-HCl, pH 8.5; Gibco). 100 µl of this culture was added to 500 µl of lysis buffer (50 mM Tris-HCl pH 8, 70 mM EDTA pH 8, 1% sodium dodecyl sulfate, Sigma), containing 0.5 µg/ml pronase (Roche, Mannheim, Germany) and acid washed glass beads (Sigma). After vigorous vortexing for 10 s, the samples were incubated at 37°C during 4 min. Next, 400 µl of saturated ammonium acetate was added and the samples were vortexed for 10 s prior to centrifugation (2 min, 13000 rpm). Subsequently, 600 µl of chloroform was added and the samples were vortexed for another 10 s. The phases were separated by centrifugation (5 min, 13000 rpm) and 400 µl of the top aqueous phase was transferred to a DNAse free Eppendorf tube (Lobind tube, Eppendorf, Aarschot, Belgium) containing 1 ml of 100% ethanol. After centrifugation (5 min, 13000 rpm) the pellet was washed with 500 µl of 70% ethanol and dried in a SpeedVac (Thermo Fisher Scientific, USA) for 10 min. Finally, the extracted DNA was dissolved in 100 µl of low EDTA- Tris buffer (0.1 mM EDTA, 10 mM Tris-HCl, pH 8.5) containing 0.5 µg/ml RNAse (Qiagen, Venlo, The Netherlands) and was incubated at 37°C for 1 h. The DNA was subsequently quantified using a BioDrop µLITE (BioDrop, Cambridge, UK). NEB prep kit ENA first public 2021-03-05 ENA last update 2021-02-18 External Id SAMEA8105919 INSDC center alias Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC center name Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC first public 2021-03-05T04:11:57Z INSDC last update 2021-02-18T14:20:21Z INSDC status public Submitter Id E-MTAB-10124:L2C16 control broker name ArrayExpress compound DMSO dose 0.25 genotype wild type genotype growth condition SCFM2 individual 2 organism part whole organism passage 16 sample name E-MTAB-10124:L2C16 control strain PAO1 ERS5792994 SAMEA8105920 208964 Pseudomonas aeruginosa PAO1 Protocols: The biofilm is treated for 24h with tobramycin and C-30 alone or a combination of both for 24h. A sample of the treated biofilm is inoculated in LB and incubated overnight. The DNA-extraction is performed on 3ml of this overnight culture with OD590 = 2. P. aeruginosa PAO1 biofilms were grown in the synthetic cystic fibrosis sputum medium (SCFM2) for 24h. The biofilm was grown in a flat bottom 96-well plate and the inoculum was approx. 5 x 107 CFU/ml. P. aeruginosa biofilms were treated with tobramycin, furanone C-30 or a combination of both in fresh SCFM2 to obtain a final concentration of 20 µg/ml tobramycin and 100 µg/ml furanone C-30. The negative control was treated with fresh SCFM2, including the same amount of DMSO (0.25%) as for the biofilms treated with C-30. After 24 h of static incubation at 37°C, biofilms were sonicated and vortexed in order to disintegrate the biofilm aggregates. A sample from this biofilm was used to start a new cycle. For each treatment three independent lineages were established, that were each exposed for 16 cycles. A sample from the treated biofilm was regrown overnight in LB and 3 ml of a culture with OD590 = 2 was resuspended in 200 µl Tris- EDTA buffer (1mM EDTA, 10 mM Tris-HCl, pH 8.5; Gibco). 100 µl of this culture was added to 500 µl of lysis buffer (50 mM Tris-HCl pH 8, 70 mM EDTA pH 8, 1% sodium dodecyl sulfate, Sigma), containing 0.5 µg/ml pronase (Roche, Mannheim, Germany) and acid washed glass beads (Sigma). After vigorous vortexing for 10 s, the samples were incubated at 37°C during 4 min. Next, 400 µl of saturated ammonium acetate was added and the samples were vortexed for 10 s prior to centrifugation (2 min, 13000 rpm). Subsequently, 600 µl of chloroform was added and the samples were vortexed for another 10 s. The phases were separated by centrifugation (5 min, 13000 rpm) and 400 µl of the top aqueous phase was transferred to a DNAse free Eppendorf tube (Lobind tube, Eppendorf, Aarschot, Belgium) containing 1 ml of 100% ethanol. After centrifugation (5 min, 13000 rpm) the pellet was washed with 500 µl of 70% ethanol and dried in a SpeedVac (Thermo Fisher Scientific, USA) for 10 min. Finally, the extracted DNA was dissolved in 100 µl of low EDTA- Tris buffer (0.1 mM EDTA, 10 mM Tris-HCl, pH 8.5) containing 0.5 µg/ml RNAse (Qiagen, Venlo, The Netherlands) and was incubated at 37°C for 1 h. The DNA was subsequently quantified using a BioDrop µLITE (BioDrop, Cambridge, UK). NEB prep kit ENA first public 2021-03-05 ENA last update 2021-02-18 External Id SAMEA8105920 INSDC center alias Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC center name Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC first public 2021-03-05T04:11:57Z INSDC last update 2021-02-18T14:20:21Z INSDC status public Submitter Id E-MTAB-10124:L2C16 tob broker name ArrayExpress compound tobramycin dose 20 genotype wild type genotype growth condition SCFM2 individual 2 organism part whole organism passage 16 sample name E-MTAB-10124:L2C16 tob strain PAO1 ERS5792995 SAMEA8105921 208964 Pseudomonas aeruginosa PAO1 Protocols: The biofilm is treated for 24h with tobramycin and C-30 alone or a combination of both for 24h. A sample of the treated biofilm is inoculated in LB and incubated overnight. The DNA-extraction is performed on 3ml of this overnight culture with OD590 = 2. P. aeruginosa PAO1 biofilms were grown in the synthetic cystic fibrosis sputum medium (SCFM2) for 24h. The biofilm was grown in a flat bottom 96-well plate and the inoculum was approx. 5 x 107 CFU/ml. P. aeruginosa biofilms were treated with tobramycin, furanone C-30 or a combination of both in fresh SCFM2 to obtain a final concentration of 20 µg/ml tobramycin and 100 µg/ml furanone C-30. The negative control was treated with fresh SCFM2, including the same amount of DMSO (0.25%) as for the biofilms treated with C-30. After 24 h of static incubation at 37°C, biofilms were sonicated and vortexed in order to disintegrate the biofilm aggregates. A sample from this biofilm was used to start a new cycle. For each treatment three independent lineages were established, that were each exposed for 16 cycles. A sample from the treated biofilm was regrown overnight in LB and 3 ml of a culture with OD590 = 2 was resuspended in 200 µl Tris- EDTA buffer (1mM EDTA, 10 mM Tris-HCl, pH 8.5; Gibco). 100 µl of this culture was added to 500 µl of lysis buffer (50 mM Tris-HCl pH 8, 70 mM EDTA pH 8, 1% sodium dodecyl sulfate, Sigma), containing 0.5 µg/ml pronase (Roche, Mannheim, Germany) and acid washed glass beads (Sigma). After vigorous vortexing for 10 s, the samples were incubated at 37°C during 4 min. Next, 400 µl of saturated ammonium acetate was added and the samples were vortexed for 10 s prior to centrifugation (2 min, 13000 rpm). Subsequently, 600 µl of chloroform was added and the samples were vortexed for another 10 s. The phases were separated by centrifugation (5 min, 13000 rpm) and 400 µl of the top aqueous phase was transferred to a DNAse free Eppendorf tube (Lobind tube, Eppendorf, Aarschot, Belgium) containing 1 ml of 100% ethanol. After centrifugation (5 min, 13000 rpm) the pellet was washed with 500 µl of 70% ethanol and dried in a SpeedVac (Thermo Fisher Scientific, USA) for 10 min. Finally, the extracted DNA was dissolved in 100 µl of low EDTA- Tris buffer (0.1 mM EDTA, 10 mM Tris-HCl, pH 8.5) containing 0.5 µg/ml RNAse (Qiagen, Venlo, The Netherlands) and was incubated at 37°C for 1 h. The DNA was subsequently quantified using a BioDrop µLITE (BioDrop, Cambridge, UK). NEB prep kit ENA first public 2021-03-05 ENA last update 2021-02-18 External Id SAMEA8105921 INSDC center alias Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC center name Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC first public 2021-03-05T04:11:57Z INSDC last update 2021-02-18T14:20:21Z INSDC status public Submitter Id E-MTAB-10124:L2C16 tob + C30 broker name ArrayExpress compound tobramycin; 20 microgram per mililiter and DMSO; 0.25 percent genotype wild type genotype growth condition SCFM2 individual 2 organism part whole organism passage 16 sample name E-MTAB-10124:L2C16 tob + C30 stimulus furanone C-30; 100 microgram per mililiter strain PAO1 ERS5792996 SAMEA8105922 208964 Pseudomonas aeruginosa PAO1 Protocols: The biofilm is treated for 24h with tobramycin and C-30 alone or a combination of both for 24h. A sample of the treated biofilm is inoculated in LB and incubated overnight. The DNA-extraction is performed on 3ml of this overnight culture with OD590 = 2. P. aeruginosa PAO1 biofilms were grown in the synthetic cystic fibrosis sputum medium (SCFM2) for 24h. The biofilm was grown in a flat bottom 96-well plate and the inoculum was approx. 5 x 107 CFU/ml. P. aeruginosa biofilms were treated with tobramycin, furanone C-30 or a combination of both in fresh SCFM2 to obtain a final concentration of 20 µg/ml tobramycin and 100 µg/ml furanone C-30. The negative control was treated with fresh SCFM2, including the same amount of DMSO (0.25%) as for the biofilms treated with C-30. After 24 h of static incubation at 37°C, biofilms were sonicated and vortexed in order to disintegrate the biofilm aggregates. A sample from this biofilm was used to start a new cycle. For each treatment three independent lineages were established, that were each exposed for 16 cycles. A sample from the treated biofilm was regrown overnight in LB and 3 ml of a culture with OD590 = 2 was resuspended in 200 µl Tris- EDTA buffer (1mM EDTA, 10 mM Tris-HCl, pH 8.5; Gibco). 100 µl of this culture was added to 500 µl of lysis buffer (50 mM Tris-HCl pH 8, 70 mM EDTA pH 8, 1% sodium dodecyl sulfate, Sigma), containing 0.5 µg/ml pronase (Roche, Mannheim, Germany) and acid washed glass beads (Sigma). After vigorous vortexing for 10 s, the samples were incubated at 37°C during 4 min. Next, 400 µl of saturated ammonium acetate was added and the samples were vortexed for 10 s prior to centrifugation (2 min, 13000 rpm). Subsequently, 600 µl of chloroform was added and the samples were vortexed for another 10 s. The phases were separated by centrifugation (5 min, 13000 rpm) and 400 µl of the top aqueous phase was transferred to a DNAse free Eppendorf tube (Lobind tube, Eppendorf, Aarschot, Belgium) containing 1 ml of 100% ethanol. After centrifugation (5 min, 13000 rpm) the pellet was washed with 500 µl of 70% ethanol and dried in a SpeedVac (Thermo Fisher Scientific, USA) for 10 min. Finally, the extracted DNA was dissolved in 100 µl of low EDTA- Tris buffer (0.1 mM EDTA, 10 mM Tris-HCl, pH 8.5) containing 0.5 µg/ml RNAse (Qiagen, Venlo, The Netherlands) and was incubated at 37°C for 1 h. The DNA was subsequently quantified using a BioDrop µLITE (BioDrop, Cambridge, UK). NEB prep kit ENA first public 2021-03-05 ENA last update 2021-02-18 External Id SAMEA8105922 INSDC center alias Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC center name Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC first public 2021-03-05T04:11:57Z INSDC last update 2021-02-18T14:20:21Z INSDC status public Submitter Id E-MTAB-10124:L2C5 C30 broker name ArrayExpress compound DMSO dose 0.25 genotype wild type genotype growth condition SCFM2 individual 2 organism part whole organism passage 5 sample name E-MTAB-10124:L2C5 C30 stimulus furanone C-30; 100 microgram per mililiter strain PAO1 ERS5792997 SAMEA8105923 208964 Pseudomonas aeruginosa PAO1 Protocols: The biofilm is treated for 24h with tobramycin and C-30 alone or a combination of both for 24h. A sample of the treated biofilm is inoculated in LB and incubated overnight. The DNA-extraction is performed on 3ml of this overnight culture with OD590 = 2. P. aeruginosa PAO1 biofilms were grown in the synthetic cystic fibrosis sputum medium (SCFM2) for 24h. The biofilm was grown in a flat bottom 96-well plate and the inoculum was approx. 5 x 107 CFU/ml. P. aeruginosa biofilms were treated with tobramycin, furanone C-30 or a combination of both in fresh SCFM2 to obtain a final concentration of 20 µg/ml tobramycin and 100 µg/ml furanone C-30. The negative control was treated with fresh SCFM2, including the same amount of DMSO (0.25%) as for the biofilms treated with C-30. After 24 h of static incubation at 37°C, biofilms were sonicated and vortexed in order to disintegrate the biofilm aggregates. A sample from this biofilm was used to start a new cycle. For each treatment three independent lineages were established, that were each exposed for 16 cycles. A sample from the treated biofilm was regrown overnight in LB and 3 ml of a culture with OD590 = 2 was resuspended in 200 µl Tris- EDTA buffer (1mM EDTA, 10 mM Tris-HCl, pH 8.5; Gibco). 100 µl of this culture was added to 500 µl of lysis buffer (50 mM Tris-HCl pH 8, 70 mM EDTA pH 8, 1% sodium dodecyl sulfate, Sigma), containing 0.5 µg/ml pronase (Roche, Mannheim, Germany) and acid washed glass beads (Sigma). After vigorous vortexing for 10 s, the samples were incubated at 37°C during 4 min. Next, 400 µl of saturated ammonium acetate was added and the samples were vortexed for 10 s prior to centrifugation (2 min, 13000 rpm). Subsequently, 600 µl of chloroform was added and the samples were vortexed for another 10 s. The phases were separated by centrifugation (5 min, 13000 rpm) and 400 µl of the top aqueous phase was transferred to a DNAse free Eppendorf tube (Lobind tube, Eppendorf, Aarschot, Belgium) containing 1 ml of 100% ethanol. After centrifugation (5 min, 13000 rpm) the pellet was washed with 500 µl of 70% ethanol and dried in a SpeedVac (Thermo Fisher Scientific, USA) for 10 min. Finally, the extracted DNA was dissolved in 100 µl of low EDTA- Tris buffer (0.1 mM EDTA, 10 mM Tris-HCl, pH 8.5) containing 0.5 µg/ml RNAse (Qiagen, Venlo, The Netherlands) and was incubated at 37°C for 1 h. The DNA was subsequently quantified using a BioDrop µLITE (BioDrop, Cambridge, UK). NEB prep kit ENA first public 2021-03-05 ENA last update 2021-02-18 External Id SAMEA8105923 INSDC center alias Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC center name Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC first public 2021-03-05T04:11:57Z INSDC last update 2021-02-18T14:20:21Z INSDC status public Submitter Id E-MTAB-10124:L2C5 control broker name ArrayExpress compound DMSO dose 0.25 genotype wild type genotype growth condition SCFM2 individual 2 organism part whole organism passage 5 sample name E-MTAB-10124:L2C5 control strain PAO1 ERS5792998 SAMEA8105924 208964 Pseudomonas aeruginosa PAO1 Protocols: The biofilm is treated for 24h with tobramycin and C-30 alone or a combination of both for 24h. A sample of the treated biofilm is inoculated in LB and incubated overnight. The DNA-extraction is performed on 3ml of this overnight culture with OD590 = 2. P. aeruginosa PAO1 biofilms were grown in the synthetic cystic fibrosis sputum medium (SCFM2) for 24h. The biofilm was grown in a flat bottom 96-well plate and the inoculum was approx. 5 x 107 CFU/ml. P. aeruginosa biofilms were treated with tobramycin, furanone C-30 or a combination of both in fresh SCFM2 to obtain a final concentration of 20 µg/ml tobramycin and 100 µg/ml furanone C-30. The negative control was treated with fresh SCFM2, including the same amount of DMSO (0.25%) as for the biofilms treated with C-30. After 24 h of static incubation at 37°C, biofilms were sonicated and vortexed in order to disintegrate the biofilm aggregates. A sample from this biofilm was used to start a new cycle. For each treatment three independent lineages were established, that were each exposed for 16 cycles. A sample from the treated biofilm was regrown overnight in LB and 3 ml of a culture with OD590 = 2 was resuspended in 200 µl Tris- EDTA buffer (1mM EDTA, 10 mM Tris-HCl, pH 8.5; Gibco). 100 µl of this culture was added to 500 µl of lysis buffer (50 mM Tris-HCl pH 8, 70 mM EDTA pH 8, 1% sodium dodecyl sulfate, Sigma), containing 0.5 µg/ml pronase (Roche, Mannheim, Germany) and acid washed glass beads (Sigma). After vigorous vortexing for 10 s, the samples were incubated at 37°C during 4 min. Next, 400 µl of saturated ammonium acetate was added and the samples were vortexed for 10 s prior to centrifugation (2 min, 13000 rpm). Subsequently, 600 µl of chloroform was added and the samples were vortexed for another 10 s. The phases were separated by centrifugation (5 min, 13000 rpm) and 400 µl of the top aqueous phase was transferred to a DNAse free Eppendorf tube (Lobind tube, Eppendorf, Aarschot, Belgium) containing 1 ml of 100% ethanol. After centrifugation (5 min, 13000 rpm) the pellet was washed with 500 µl of 70% ethanol and dried in a SpeedVac (Thermo Fisher Scientific, USA) for 10 min. Finally, the extracted DNA was dissolved in 100 µl of low EDTA- Tris buffer (0.1 mM EDTA, 10 mM Tris-HCl, pH 8.5) containing 0.5 µg/ml RNAse (Qiagen, Venlo, The Netherlands) and was incubated at 37°C for 1 h. The DNA was subsequently quantified using a BioDrop µLITE (BioDrop, Cambridge, UK). NEB prep kit ENA first public 2021-03-05 ENA last update 2021-02-18 External Id SAMEA8105924 INSDC center alias Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC center name Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC first public 2021-03-05T04:11:57Z INSDC last update 2021-02-18T14:20:21Z INSDC status public Submitter Id E-MTAB-10124:L2C5 tob broker name ArrayExpress compound tobramycin dose 20 genotype wild type genotype growth condition SCFM2 individual 2 organism part whole organism passage 5 sample name E-MTAB-10124:L2C5 tob strain PAO1 ERS5792999 SAMEA8105925 208964 Pseudomonas aeruginosa PAO1 Protocols: The biofilm is treated for 24h with tobramycin and C-30 alone or a combination of both for 24h. A sample of the treated biofilm is inoculated in LB and incubated overnight. The DNA-extraction is performed on 3ml of this overnight culture with OD590 = 2. P. aeruginosa PAO1 biofilms were grown in the synthetic cystic fibrosis sputum medium (SCFM2) for 24h. The biofilm was grown in a flat bottom 96-well plate and the inoculum was approx. 5 x 107 CFU/ml. P. aeruginosa biofilms were treated with tobramycin, furanone C-30 or a combination of both in fresh SCFM2 to obtain a final concentration of 20 µg/ml tobramycin and 100 µg/ml furanone C-30. The negative control was treated with fresh SCFM2, including the same amount of DMSO (0.25%) as for the biofilms treated with C-30. After 24 h of static incubation at 37°C, biofilms were sonicated and vortexed in order to disintegrate the biofilm aggregates. A sample from this biofilm was used to start a new cycle. For each treatment three independent lineages were established, that were each exposed for 16 cycles. A sample from the treated biofilm was regrown overnight in LB and 3 ml of a culture with OD590 = 2 was resuspended in 200 µl Tris- EDTA buffer (1mM EDTA, 10 mM Tris-HCl, pH 8.5; Gibco). 100 µl of this culture was added to 500 µl of lysis buffer (50 mM Tris-HCl pH 8, 70 mM EDTA pH 8, 1% sodium dodecyl sulfate, Sigma), containing 0.5 µg/ml pronase (Roche, Mannheim, Germany) and acid washed glass beads (Sigma). After vigorous vortexing for 10 s, the samples were incubated at 37°C during 4 min. Next, 400 µl of saturated ammonium acetate was added and the samples were vortexed for 10 s prior to centrifugation (2 min, 13000 rpm). Subsequently, 600 µl of chloroform was added and the samples were vortexed for another 10 s. The phases were separated by centrifugation (5 min, 13000 rpm) and 400 µl of the top aqueous phase was transferred to a DNAse free Eppendorf tube (Lobind tube, Eppendorf, Aarschot, Belgium) containing 1 ml of 100% ethanol. After centrifugation (5 min, 13000 rpm) the pellet was washed with 500 µl of 70% ethanol and dried in a SpeedVac (Thermo Fisher Scientific, USA) for 10 min. Finally, the extracted DNA was dissolved in 100 µl of low EDTA- Tris buffer (0.1 mM EDTA, 10 mM Tris-HCl, pH 8.5) containing 0.5 µg/ml RNAse (Qiagen, Venlo, The Netherlands) and was incubated at 37°C for 1 h. The DNA was subsequently quantified using a BioDrop µLITE (BioDrop, Cambridge, UK). NEB prep kit ENA first public 2021-03-05 ENA last update 2021-02-18 External Id SAMEA8105925 INSDC center alias Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC center name Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC first public 2021-03-05T04:11:57Z INSDC last update 2021-02-18T14:20:21Z INSDC status public Submitter Id E-MTAB-10124:L2C5 tob + C30 broker name ArrayExpress compound tobramycin; 20 microgram per mililiter and DMSO; 0.25 percent genotype wild type genotype growth condition SCFM2 individual 2 organism part whole organism passage 5 sample name E-MTAB-10124:L2C5 tob + C30 stimulus furanone C-30; 100 microgram per mililiter strain PAO1 ERS5793000 SAMEA8105926 208964 Pseudomonas aeruginosa PAO1 Protocols: The biofilm is treated for 24h with tobramycin and C-30 alone or a combination of both for 24h. A sample of the treated biofilm is inoculated in LB and incubated overnight. The DNA-extraction is performed on 3ml of this overnight culture with OD590 = 2. P. aeruginosa PAO1 biofilms were grown in the synthetic cystic fibrosis sputum medium (SCFM2) for 24h. The biofilm was grown in a flat bottom 96-well plate and the inoculum was approx. 5 x 107 CFU/ml. P. aeruginosa biofilms were treated with tobramycin, furanone C-30 or a combination of both in fresh SCFM2 to obtain a final concentration of 20 µg/ml tobramycin and 100 µg/ml furanone C-30. The negative control was treated with fresh SCFM2, including the same amount of DMSO (0.25%) as for the biofilms treated with C-30. After 24 h of static incubation at 37°C, biofilms were sonicated and vortexed in order to disintegrate the biofilm aggregates. A sample from this biofilm was used to start a new cycle. For each treatment three independent lineages were established, that were each exposed for 16 cycles. A sample from the treated biofilm was regrown overnight in LB and 3 ml of a culture with OD590 = 2 was resuspended in 200 µl Tris- EDTA buffer (1mM EDTA, 10 mM Tris-HCl, pH 8.5; Gibco). 100 µl of this culture was added to 500 µl of lysis buffer (50 mM Tris-HCl pH 8, 70 mM EDTA pH 8, 1% sodium dodecyl sulfate, Sigma), containing 0.5 µg/ml pronase (Roche, Mannheim, Germany) and acid washed glass beads (Sigma). After vigorous vortexing for 10 s, the samples were incubated at 37°C during 4 min. Next, 400 µl of saturated ammonium acetate was added and the samples were vortexed for 10 s prior to centrifugation (2 min, 13000 rpm). Subsequently, 600 µl of chloroform was added and the samples were vortexed for another 10 s. The phases were separated by centrifugation (5 min, 13000 rpm) and 400 µl of the top aqueous phase was transferred to a DNAse free Eppendorf tube (Lobind tube, Eppendorf, Aarschot, Belgium) containing 1 ml of 100% ethanol. After centrifugation (5 min, 13000 rpm) the pellet was washed with 500 µl of 70% ethanol and dried in a SpeedVac (Thermo Fisher Scientific, USA) for 10 min. Finally, the extracted DNA was dissolved in 100 µl of low EDTA- Tris buffer (0.1 mM EDTA, 10 mM Tris-HCl, pH 8.5) containing 0.5 µg/ml RNAse (Qiagen, Venlo, The Netherlands) and was incubated at 37°C for 1 h. The DNA was subsequently quantified using a BioDrop µLITE (BioDrop, Cambridge, UK). NEB prep kit ENA first public 2021-03-05 ENA last update 2021-02-18 External Id SAMEA8105926 INSDC center alias Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC center name Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC first public 2021-03-05T04:11:57Z INSDC last update 2021-02-18T14:20:21Z INSDC status public Submitter Id E-MTAB-10124:L3C10 C30 broker name ArrayExpress compound DMSO dose 0.25 genotype wild type genotype growth condition SCFM2 individual 3 organism part whole organism passage 10 sample name E-MTAB-10124:L3C10 C30 stimulus furanone C-30; 100 microgram per mililiter strain PAO1 ERS5793001 SAMEA8105927 208964 Pseudomonas aeruginosa PAO1 Protocols: The biofilm is treated for 24h with tobramycin and C-30 alone or a combination of both for 24h. A sample of the treated biofilm is inoculated in LB and incubated overnight. The DNA-extraction is performed on 3ml of this overnight culture with OD590 = 2. P. aeruginosa PAO1 biofilms were grown in the synthetic cystic fibrosis sputum medium (SCFM2) for 24h. The biofilm was grown in a flat bottom 96-well plate and the inoculum was approx. 5 x 107 CFU/ml. P. aeruginosa biofilms were treated with tobramycin, furanone C-30 or a combination of both in fresh SCFM2 to obtain a final concentration of 20 µg/ml tobramycin and 100 µg/ml furanone C-30. The negative control was treated with fresh SCFM2, including the same amount of DMSO (0.25%) as for the biofilms treated with C-30. After 24 h of static incubation at 37°C, biofilms were sonicated and vortexed in order to disintegrate the biofilm aggregates. A sample from this biofilm was used to start a new cycle. For each treatment three independent lineages were established, that were each exposed for 16 cycles. A sample from the treated biofilm was regrown overnight in LB and 3 ml of a culture with OD590 = 2 was resuspended in 200 µl Tris- EDTA buffer (1mM EDTA, 10 mM Tris-HCl, pH 8.5; Gibco). 100 µl of this culture was added to 500 µl of lysis buffer (50 mM Tris-HCl pH 8, 70 mM EDTA pH 8, 1% sodium dodecyl sulfate, Sigma), containing 0.5 µg/ml pronase (Roche, Mannheim, Germany) and acid washed glass beads (Sigma). After vigorous vortexing for 10 s, the samples were incubated at 37°C during 4 min. Next, 400 µl of saturated ammonium acetate was added and the samples were vortexed for 10 s prior to centrifugation (2 min, 13000 rpm). Subsequently, 600 µl of chloroform was added and the samples were vortexed for another 10 s. The phases were separated by centrifugation (5 min, 13000 rpm) and 400 µl of the top aqueous phase was transferred to a DNAse free Eppendorf tube (Lobind tube, Eppendorf, Aarschot, Belgium) containing 1 ml of 100% ethanol. After centrifugation (5 min, 13000 rpm) the pellet was washed with 500 µl of 70% ethanol and dried in a SpeedVac (Thermo Fisher Scientific, USA) for 10 min. Finally, the extracted DNA was dissolved in 100 µl of low EDTA- Tris buffer (0.1 mM EDTA, 10 mM Tris-HCl, pH 8.5) containing 0.5 µg/ml RNAse (Qiagen, Venlo, The Netherlands) and was incubated at 37°C for 1 h. The DNA was subsequently quantified using a BioDrop µLITE (BioDrop, Cambridge, UK). NEB prep kit ENA first public 2021-03-05 ENA last update 2021-02-18 External Id SAMEA8105927 INSDC center alias Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC center name Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC first public 2021-03-05T04:11:57Z INSDC last update 2021-02-18T14:20:21Z INSDC status public Submitter Id E-MTAB-10124:L3C10 control broker name ArrayExpress compound DMSO dose 0.25 genotype wild type genotype growth condition SCFM2 individual 3 organism part whole organism passage 10 sample name E-MTAB-10124:L3C10 control strain PAO1 ERS5793002 SAMEA8105928 208964 Pseudomonas aeruginosa PAO1 Protocols: The biofilm is treated for 24h with tobramycin and C-30 alone or a combination of both for 24h. A sample of the treated biofilm is inoculated in LB and incubated overnight. The DNA-extraction is performed on 3ml of this overnight culture with OD590 = 2. P. aeruginosa PAO1 biofilms were grown in the synthetic cystic fibrosis sputum medium (SCFM2) for 24h. The biofilm was grown in a flat bottom 96-well plate and the inoculum was approx. 5 x 107 CFU/ml. P. aeruginosa biofilms were treated with tobramycin, furanone C-30 or a combination of both in fresh SCFM2 to obtain a final concentration of 20 µg/ml tobramycin and 100 µg/ml furanone C-30. The negative control was treated with fresh SCFM2, including the same amount of DMSO (0.25%) as for the biofilms treated with C-30. After 24 h of static incubation at 37°C, biofilms were sonicated and vortexed in order to disintegrate the biofilm aggregates. A sample from this biofilm was used to start a new cycle. For each treatment three independent lineages were established, that were each exposed for 16 cycles. A sample from the treated biofilm was regrown overnight in LB and 3 ml of a culture with OD590 = 2 was resuspended in 200 µl Tris- EDTA buffer (1mM EDTA, 10 mM Tris-HCl, pH 8.5; Gibco). 100 µl of this culture was added to 500 µl of lysis buffer (50 mM Tris-HCl pH 8, 70 mM EDTA pH 8, 1% sodium dodecyl sulfate, Sigma), containing 0.5 µg/ml pronase (Roche, Mannheim, Germany) and acid washed glass beads (Sigma). After vigorous vortexing for 10 s, the samples were incubated at 37°C during 4 min. Next, 400 µl of saturated ammonium acetate was added and the samples were vortexed for 10 s prior to centrifugation (2 min, 13000 rpm). Subsequently, 600 µl of chloroform was added and the samples were vortexed for another 10 s. The phases were separated by centrifugation (5 min, 13000 rpm) and 400 µl of the top aqueous phase was transferred to a DNAse free Eppendorf tube (Lobind tube, Eppendorf, Aarschot, Belgium) containing 1 ml of 100% ethanol. After centrifugation (5 min, 13000 rpm) the pellet was washed with 500 µl of 70% ethanol and dried in a SpeedVac (Thermo Fisher Scientific, USA) for 10 min. Finally, the extracted DNA was dissolved in 100 µl of low EDTA- Tris buffer (0.1 mM EDTA, 10 mM Tris-HCl, pH 8.5) containing 0.5 µg/ml RNAse (Qiagen, Venlo, The Netherlands) and was incubated at 37°C for 1 h. The DNA was subsequently quantified using a BioDrop µLITE (BioDrop, Cambridge, UK). NEB prep kit ENA first public 2021-03-05 ENA last update 2021-02-18 External Id SAMEA8105928 INSDC center alias Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC center name Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC first public 2021-03-05T04:11:57Z INSDC last update 2021-02-18T14:20:21Z INSDC status public Submitter Id E-MTAB-10124:L3C10 tob broker name ArrayExpress compound tobramycin dose 20 genotype wild type genotype growth condition SCFM2 individual 3 organism part whole organism passage 10 sample name E-MTAB-10124:L3C10 tob strain PAO1 ERS5793003 SAMEA8105929 208964 Pseudomonas aeruginosa PAO1 Protocols: The biofilm is treated for 24h with tobramycin and C-30 alone or a combination of both for 24h. A sample of the treated biofilm is inoculated in LB and incubated overnight. The DNA-extraction is performed on 3ml of this overnight culture with OD590 = 2. P. aeruginosa PAO1 biofilms were grown in the synthetic cystic fibrosis sputum medium (SCFM2) for 24h. The biofilm was grown in a flat bottom 96-well plate and the inoculum was approx. 5 x 107 CFU/ml. P. aeruginosa biofilms were treated with tobramycin, furanone C-30 or a combination of both in fresh SCFM2 to obtain a final concentration of 20 µg/ml tobramycin and 100 µg/ml furanone C-30. The negative control was treated with fresh SCFM2, including the same amount of DMSO (0.25%) as for the biofilms treated with C-30. After 24 h of static incubation at 37°C, biofilms were sonicated and vortexed in order to disintegrate the biofilm aggregates. A sample from this biofilm was used to start a new cycle. For each treatment three independent lineages were established, that were each exposed for 16 cycles. A sample from the treated biofilm was regrown overnight in LB and 3 ml of a culture with OD590 = 2 was resuspended in 200 µl Tris- EDTA buffer (1mM EDTA, 10 mM Tris-HCl, pH 8.5; Gibco). 100 µl of this culture was added to 500 µl of lysis buffer (50 mM Tris-HCl pH 8, 70 mM EDTA pH 8, 1% sodium dodecyl sulfate, Sigma), containing 0.5 µg/ml pronase (Roche, Mannheim, Germany) and acid washed glass beads (Sigma). After vigorous vortexing for 10 s, the samples were incubated at 37°C during 4 min. Next, 400 µl of saturated ammonium acetate was added and the samples were vortexed for 10 s prior to centrifugation (2 min, 13000 rpm). Subsequently, 600 µl of chloroform was added and the samples were vortexed for another 10 s. The phases were separated by centrifugation (5 min, 13000 rpm) and 400 µl of the top aqueous phase was transferred to a DNAse free Eppendorf tube (Lobind tube, Eppendorf, Aarschot, Belgium) containing 1 ml of 100% ethanol. After centrifugation (5 min, 13000 rpm) the pellet was washed with 500 µl of 70% ethanol and dried in a SpeedVac (Thermo Fisher Scientific, USA) for 10 min. Finally, the extracted DNA was dissolved in 100 µl of low EDTA- Tris buffer (0.1 mM EDTA, 10 mM Tris-HCl, pH 8.5) containing 0.5 µg/ml RNAse (Qiagen, Venlo, The Netherlands) and was incubated at 37°C for 1 h. The DNA was subsequently quantified using a BioDrop µLITE (BioDrop, Cambridge, UK). NEB prep kit ENA first public 2021-03-05 ENA last update 2021-02-18 External Id SAMEA8105929 INSDC center alias Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC center name Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC first public 2021-03-05T04:11:57Z INSDC last update 2021-02-18T14:20:21Z INSDC status public Submitter Id E-MTAB-10124:L3C10 tob + C30 broker name ArrayExpress compound tobramycin; 20 microgram per mililiter and DMSO; 0.25 percent genotype wild type genotype growth condition SCFM2 individual 3 organism part whole organism passage 10 sample name E-MTAB-10124:L3C10 tob + C30 stimulus furanone C-30; 100 microgram per mililiter strain PAO1 ERS5793004 SAMEA8105930 208964 Pseudomonas aeruginosa PAO1 Protocols: The biofilm is treated for 24h with tobramycin and C-30 alone or a combination of both for 24h. A sample of the treated biofilm is inoculated in LB and incubated overnight. The DNA-extraction is performed on 3ml of this overnight culture with OD590 = 2. P. aeruginosa PAO1 biofilms were grown in the synthetic cystic fibrosis sputum medium (SCFM2) for 24h. The biofilm was grown in a flat bottom 96-well plate and the inoculum was approx. 5 x 107 CFU/ml. P. aeruginosa biofilms were treated with tobramycin, furanone C-30 or a combination of both in fresh SCFM2 to obtain a final concentration of 20 µg/ml tobramycin and 100 µg/ml furanone C-30. The negative control was treated with fresh SCFM2, including the same amount of DMSO (0.25%) as for the biofilms treated with C-30. After 24 h of static incubation at 37°C, biofilms were sonicated and vortexed in order to disintegrate the biofilm aggregates. A sample from this biofilm was used to start a new cycle. For each treatment three independent lineages were established, that were each exposed for 16 cycles. A sample from the treated biofilm was regrown overnight in LB and 3 ml of a culture with OD590 = 2 was resuspended in 200 µl Tris- EDTA buffer (1mM EDTA, 10 mM Tris-HCl, pH 8.5; Gibco). 100 µl of this culture was added to 500 µl of lysis buffer (50 mM Tris-HCl pH 8, 70 mM EDTA pH 8, 1% sodium dodecyl sulfate, Sigma), containing 0.5 µg/ml pronase (Roche, Mannheim, Germany) and acid washed glass beads (Sigma). After vigorous vortexing for 10 s, the samples were incubated at 37°C during 4 min. Next, 400 µl of saturated ammonium acetate was added and the samples were vortexed for 10 s prior to centrifugation (2 min, 13000 rpm). Subsequently, 600 µl of chloroform was added and the samples were vortexed for another 10 s. The phases were separated by centrifugation (5 min, 13000 rpm) and 400 µl of the top aqueous phase was transferred to a DNAse free Eppendorf tube (Lobind tube, Eppendorf, Aarschot, Belgium) containing 1 ml of 100% ethanol. After centrifugation (5 min, 13000 rpm) the pellet was washed with 500 µl of 70% ethanol and dried in a SpeedVac (Thermo Fisher Scientific, USA) for 10 min. Finally, the extracted DNA was dissolved in 100 µl of low EDTA- Tris buffer (0.1 mM EDTA, 10 mM Tris-HCl, pH 8.5) containing 0.5 µg/ml RNAse (Qiagen, Venlo, The Netherlands) and was incubated at 37°C for 1 h. The DNA was subsequently quantified using a BioDrop µLITE (BioDrop, Cambridge, UK). NEB prep kit ENA first public 2021-03-05 ENA last update 2021-02-18 External Id SAMEA8105930 INSDC center alias Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC center name Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC first public 2021-03-05T04:11:57Z INSDC last update 2021-02-18T14:20:21Z INSDC status public Submitter Id E-MTAB-10124:L3C16 C30 broker name ArrayExpress compound DMSO dose 0.25 genotype wild type genotype growth condition SCFM2 individual 3 organism part whole organism passage 16 sample name E-MTAB-10124:L3C16 C30 stimulus furanone C-30; 100 microgram per mililiter strain PAO1 ERS5793005 SAMEA8105931 208964 Pseudomonas aeruginosa PAO1 Protocols: The biofilm is treated for 24h with tobramycin and C-30 alone or a combination of both for 24h. A sample of the treated biofilm is inoculated in LB and incubated overnight. The DNA-extraction is performed on 3ml of this overnight culture with OD590 = 2. P. aeruginosa PAO1 biofilms were grown in the synthetic cystic fibrosis sputum medium (SCFM2) for 24h. The biofilm was grown in a flat bottom 96-well plate and the inoculum was approx. 5 x 107 CFU/ml. P. aeruginosa biofilms were treated with tobramycin, furanone C-30 or a combination of both in fresh SCFM2 to obtain a final concentration of 20 µg/ml tobramycin and 100 µg/ml furanone C-30. The negative control was treated with fresh SCFM2, including the same amount of DMSO (0.25%) as for the biofilms treated with C-30. After 24 h of static incubation at 37°C, biofilms were sonicated and vortexed in order to disintegrate the biofilm aggregates. A sample from this biofilm was used to start a new cycle. For each treatment three independent lineages were established, that were each exposed for 16 cycles. A sample from the treated biofilm was regrown overnight in LB and 3 ml of a culture with OD590 = 2 was resuspended in 200 µl Tris- EDTA buffer (1mM EDTA, 10 mM Tris-HCl, pH 8.5; Gibco). 100 µl of this culture was added to 500 µl of lysis buffer (50 mM Tris-HCl pH 8, 70 mM EDTA pH 8, 1% sodium dodecyl sulfate, Sigma), containing 0.5 µg/ml pronase (Roche, Mannheim, Germany) and acid washed glass beads (Sigma). After vigorous vortexing for 10 s, the samples were incubated at 37°C during 4 min. Next, 400 µl of saturated ammonium acetate was added and the samples were vortexed for 10 s prior to centrifugation (2 min, 13000 rpm). Subsequently, 600 µl of chloroform was added and the samples were vortexed for another 10 s. The phases were separated by centrifugation (5 min, 13000 rpm) and 400 µl of the top aqueous phase was transferred to a DNAse free Eppendorf tube (Lobind tube, Eppendorf, Aarschot, Belgium) containing 1 ml of 100% ethanol. After centrifugation (5 min, 13000 rpm) the pellet was washed with 500 µl of 70% ethanol and dried in a SpeedVac (Thermo Fisher Scientific, USA) for 10 min. Finally, the extracted DNA was dissolved in 100 µl of low EDTA- Tris buffer (0.1 mM EDTA, 10 mM Tris-HCl, pH 8.5) containing 0.5 µg/ml RNAse (Qiagen, Venlo, The Netherlands) and was incubated at 37°C for 1 h. The DNA was subsequently quantified using a BioDrop µLITE (BioDrop, Cambridge, UK). NEB prep kit ENA first public 2021-03-05 ENA last update 2021-02-18 External Id SAMEA8105931 INSDC center alias Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC center name Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC first public 2021-03-05T04:11:57Z INSDC last update 2021-02-18T14:20:22Z INSDC status public Submitter Id E-MTAB-10124:L3C16 control broker name ArrayExpress compound DMSO dose 0.25 genotype wild type genotype growth condition SCFM2 individual 3 organism part whole organism passage 16 sample name E-MTAB-10124:L3C16 control strain PAO1 ERS5793006 SAMEA8105932 208964 Pseudomonas aeruginosa PAO1 Protocols: The biofilm is treated for 24h with tobramycin and C-30 alone or a combination of both for 24h. A sample of the treated biofilm is inoculated in LB and incubated overnight. The DNA-extraction is performed on 3ml of this overnight culture with OD590 = 2. P. aeruginosa PAO1 biofilms were grown in the synthetic cystic fibrosis sputum medium (SCFM2) for 24h. The biofilm was grown in a flat bottom 96-well plate and the inoculum was approx. 5 x 107 CFU/ml. P. aeruginosa biofilms were treated with tobramycin, furanone C-30 or a combination of both in fresh SCFM2 to obtain a final concentration of 20 µg/ml tobramycin and 100 µg/ml furanone C-30. The negative control was treated with fresh SCFM2, including the same amount of DMSO (0.25%) as for the biofilms treated with C-30. After 24 h of static incubation at 37°C, biofilms were sonicated and vortexed in order to disintegrate the biofilm aggregates. A sample from this biofilm was used to start a new cycle. For each treatment three independent lineages were established, that were each exposed for 16 cycles. A sample from the treated biofilm was regrown overnight in LB and 3 ml of a culture with OD590 = 2 was resuspended in 200 µl Tris- EDTA buffer (1mM EDTA, 10 mM Tris-HCl, pH 8.5; Gibco). 100 µl of this culture was added to 500 µl of lysis buffer (50 mM Tris-HCl pH 8, 70 mM EDTA pH 8, 1% sodium dodecyl sulfate, Sigma), containing 0.5 µg/ml pronase (Roche, Mannheim, Germany) and acid washed glass beads (Sigma). After vigorous vortexing for 10 s, the samples were incubated at 37°C during 4 min. Next, 400 µl of saturated ammonium acetate was added and the samples were vortexed for 10 s prior to centrifugation (2 min, 13000 rpm). Subsequently, 600 µl of chloroform was added and the samples were vortexed for another 10 s. The phases were separated by centrifugation (5 min, 13000 rpm) and 400 µl of the top aqueous phase was transferred to a DNAse free Eppendorf tube (Lobind tube, Eppendorf, Aarschot, Belgium) containing 1 ml of 100% ethanol. After centrifugation (5 min, 13000 rpm) the pellet was washed with 500 µl of 70% ethanol and dried in a SpeedVac (Thermo Fisher Scientific, USA) for 10 min. Finally, the extracted DNA was dissolved in 100 µl of low EDTA- Tris buffer (0.1 mM EDTA, 10 mM Tris-HCl, pH 8.5) containing 0.5 µg/ml RNAse (Qiagen, Venlo, The Netherlands) and was incubated at 37°C for 1 h. The DNA was subsequently quantified using a BioDrop µLITE (BioDrop, Cambridge, UK). NEB prep kit ENA first public 2021-03-05 ENA last update 2021-02-18 External Id SAMEA8105932 INSDC center alias Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC center name Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC first public 2021-03-05T04:11:57Z INSDC last update 2021-02-18T14:20:22Z INSDC status public Submitter Id E-MTAB-10124:L3C16 tob broker name ArrayExpress compound tobramycin dose 20 genotype wild type genotype growth condition SCFM2 individual 3 organism part whole organism passage 16 sample name E-MTAB-10124:L3C16 tob strain PAO1 ERS5793007 SAMEA8105933 208964 Pseudomonas aeruginosa PAO1 Protocols: The biofilm is treated for 24h with tobramycin and C-30 alone or a combination of both for 24h. A sample of the treated biofilm is inoculated in LB and incubated overnight. The DNA-extraction is performed on 3ml of this overnight culture with OD590 = 2. P. aeruginosa PAO1 biofilms were grown in the synthetic cystic fibrosis sputum medium (SCFM2) for 24h. The biofilm was grown in a flat bottom 96-well plate and the inoculum was approx. 5 x 107 CFU/ml. P. aeruginosa biofilms were treated with tobramycin, furanone C-30 or a combination of both in fresh SCFM2 to obtain a final concentration of 20 µg/ml tobramycin and 100 µg/ml furanone C-30. The negative control was treated with fresh SCFM2, including the same amount of DMSO (0.25%) as for the biofilms treated with C-30. After 24 h of static incubation at 37°C, biofilms were sonicated and vortexed in order to disintegrate the biofilm aggregates. A sample from this biofilm was used to start a new cycle. For each treatment three independent lineages were established, that were each exposed for 16 cycles. A sample from the treated biofilm was regrown overnight in LB and 3 ml of a culture with OD590 = 2 was resuspended in 200 µl Tris- EDTA buffer (1mM EDTA, 10 mM Tris-HCl, pH 8.5; Gibco). 100 µl of this culture was added to 500 µl of lysis buffer (50 mM Tris-HCl pH 8, 70 mM EDTA pH 8, 1% sodium dodecyl sulfate, Sigma), containing 0.5 µg/ml pronase (Roche, Mannheim, Germany) and acid washed glass beads (Sigma). After vigorous vortexing for 10 s, the samples were incubated at 37°C during 4 min. Next, 400 µl of saturated ammonium acetate was added and the samples were vortexed for 10 s prior to centrifugation (2 min, 13000 rpm). Subsequently, 600 µl of chloroform was added and the samples were vortexed for another 10 s. The phases were separated by centrifugation (5 min, 13000 rpm) and 400 µl of the top aqueous phase was transferred to a DNAse free Eppendorf tube (Lobind tube, Eppendorf, Aarschot, Belgium) containing 1 ml of 100% ethanol. After centrifugation (5 min, 13000 rpm) the pellet was washed with 500 µl of 70% ethanol and dried in a SpeedVac (Thermo Fisher Scientific, USA) for 10 min. Finally, the extracted DNA was dissolved in 100 µl of low EDTA- Tris buffer (0.1 mM EDTA, 10 mM Tris-HCl, pH 8.5) containing 0.5 µg/ml RNAse (Qiagen, Venlo, The Netherlands) and was incubated at 37°C for 1 h. The DNA was subsequently quantified using a BioDrop µLITE (BioDrop, Cambridge, UK). NEB prep kit ENA first public 2021-03-05 ENA last update 2021-02-18 External Id SAMEA8105933 INSDC center alias Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC center name Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC first public 2021-03-05T04:11:57Z INSDC last update 2021-02-18T14:20:22Z INSDC status public Submitter Id E-MTAB-10124:L3C16 tob + C30 broker name ArrayExpress compound tobramycin; 20 microgram per mililiter and DMSO; 0.25 percent genotype wild type genotype growth condition SCFM2 individual 3 organism part whole organism passage 16 sample name E-MTAB-10124:L3C16 tob + C30 stimulus furanone C-30; 100 microgram per mililiter strain PAO1 ERS5793008 SAMEA8105934 208964 Pseudomonas aeruginosa PAO1 Protocols: The biofilm is treated for 24h with tobramycin and C-30 alone or a combination of both for 24h. A sample of the treated biofilm is inoculated in LB and incubated overnight. The DNA-extraction is performed on 3ml of this overnight culture with OD590 = 2. P. aeruginosa PAO1 biofilms were grown in the synthetic cystic fibrosis sputum medium (SCFM2) for 24h. The biofilm was grown in a flat bottom 96-well plate and the inoculum was approx. 5 x 107 CFU/ml. P. aeruginosa biofilms were treated with tobramycin, furanone C-30 or a combination of both in fresh SCFM2 to obtain a final concentration of 20 µg/ml tobramycin and 100 µg/ml furanone C-30. The negative control was treated with fresh SCFM2, including the same amount of DMSO (0.25%) as for the biofilms treated with C-30. After 24 h of static incubation at 37°C, biofilms were sonicated and vortexed in order to disintegrate the biofilm aggregates. A sample from this biofilm was used to start a new cycle. For each treatment three independent lineages were established, that were each exposed for 16 cycles. A sample from the treated biofilm was regrown overnight in LB and 3 ml of a culture with OD590 = 2 was resuspended in 200 µl Tris- EDTA buffer (1mM EDTA, 10 mM Tris-HCl, pH 8.5; Gibco). 100 µl of this culture was added to 500 µl of lysis buffer (50 mM Tris-HCl pH 8, 70 mM EDTA pH 8, 1% sodium dodecyl sulfate, Sigma), containing 0.5 µg/ml pronase (Roche, Mannheim, Germany) and acid washed glass beads (Sigma). After vigorous vortexing for 10 s, the samples were incubated at 37°C during 4 min. Next, 400 µl of saturated ammonium acetate was added and the samples were vortexed for 10 s prior to centrifugation (2 min, 13000 rpm). Subsequently, 600 µl of chloroform was added and the samples were vortexed for another 10 s. The phases were separated by centrifugation (5 min, 13000 rpm) and 400 µl of the top aqueous phase was transferred to a DNAse free Eppendorf tube (Lobind tube, Eppendorf, Aarschot, Belgium) containing 1 ml of 100% ethanol. After centrifugation (5 min, 13000 rpm) the pellet was washed with 500 µl of 70% ethanol and dried in a SpeedVac (Thermo Fisher Scientific, USA) for 10 min. Finally, the extracted DNA was dissolved in 100 µl of low EDTA- Tris buffer (0.1 mM EDTA, 10 mM Tris-HCl, pH 8.5) containing 0.5 µg/ml RNAse (Qiagen, Venlo, The Netherlands) and was incubated at 37°C for 1 h. The DNA was subsequently quantified using a BioDrop µLITE (BioDrop, Cambridge, UK). NEB prep kit ENA first public 2021-03-05 ENA last update 2021-02-18 External Id SAMEA8105934 INSDC center alias Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC center name Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC first public 2021-03-05T04:11:57Z INSDC last update 2021-02-18T14:20:22Z INSDC status public Submitter Id E-MTAB-10124:L3C5 C30 broker name ArrayExpress compound DMSO dose 0.25 genotype wild type genotype growth condition SCFM2 individual 3 organism part whole organism passage 5 sample name E-MTAB-10124:L3C5 C30 stimulus furanone C-30; 100 microgram per mililiter strain PAO1 ERS5793009 SAMEA8105935 208964 Pseudomonas aeruginosa PAO1 Protocols: The biofilm is treated for 24h with tobramycin and C-30 alone or a combination of both for 24h. A sample of the treated biofilm is inoculated in LB and incubated overnight. The DNA-extraction is performed on 3ml of this overnight culture with OD590 = 2. P. aeruginosa PAO1 biofilms were grown in the synthetic cystic fibrosis sputum medium (SCFM2) for 24h. The biofilm was grown in a flat bottom 96-well plate and the inoculum was approx. 5 x 107 CFU/ml. P. aeruginosa biofilms were treated with tobramycin, furanone C-30 or a combination of both in fresh SCFM2 to obtain a final concentration of 20 µg/ml tobramycin and 100 µg/ml furanone C-30. The negative control was treated with fresh SCFM2, including the same amount of DMSO (0.25%) as for the biofilms treated with C-30. After 24 h of static incubation at 37°C, biofilms were sonicated and vortexed in order to disintegrate the biofilm aggregates. A sample from this biofilm was used to start a new cycle. For each treatment three independent lineages were established, that were each exposed for 16 cycles. A sample from the treated biofilm was regrown overnight in LB and 3 ml of a culture with OD590 = 2 was resuspended in 200 µl Tris- EDTA buffer (1mM EDTA, 10 mM Tris-HCl, pH 8.5; Gibco). 100 µl of this culture was added to 500 µl of lysis buffer (50 mM Tris-HCl pH 8, 70 mM EDTA pH 8, 1% sodium dodecyl sulfate, Sigma), containing 0.5 µg/ml pronase (Roche, Mannheim, Germany) and acid washed glass beads (Sigma). After vigorous vortexing for 10 s, the samples were incubated at 37°C during 4 min. Next, 400 µl of saturated ammonium acetate was added and the samples were vortexed for 10 s prior to centrifugation (2 min, 13000 rpm). Subsequently, 600 µl of chloroform was added and the samples were vortexed for another 10 s. The phases were separated by centrifugation (5 min, 13000 rpm) and 400 µl of the top aqueous phase was transferred to a DNAse free Eppendorf tube (Lobind tube, Eppendorf, Aarschot, Belgium) containing 1 ml of 100% ethanol. After centrifugation (5 min, 13000 rpm) the pellet was washed with 500 µl of 70% ethanol and dried in a SpeedVac (Thermo Fisher Scientific, USA) for 10 min. Finally, the extracted DNA was dissolved in 100 µl of low EDTA- Tris buffer (0.1 mM EDTA, 10 mM Tris-HCl, pH 8.5) containing 0.5 µg/ml RNAse (Qiagen, Venlo, The Netherlands) and was incubated at 37°C for 1 h. The DNA was subsequently quantified using a BioDrop µLITE (BioDrop, Cambridge, UK). NEB prep kit ENA first public 2021-03-05 ENA last update 2021-02-18 External Id SAMEA8105935 INSDC center alias Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC center name Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC first public 2021-03-05T04:11:57Z INSDC last update 2021-02-18T14:20:22Z INSDC status public Submitter Id E-MTAB-10124:L3C5 control broker name ArrayExpress compound DMSO dose 0.25 genotype wild type genotype growth condition SCFM2 individual 3 organism part whole organism passage 5 sample name E-MTAB-10124:L3C5 control strain PAO1 ERS5793010 SAMEA8105936 208964 Pseudomonas aeruginosa PAO1 Protocols: The biofilm is treated for 24h with tobramycin and C-30 alone or a combination of both for 24h. A sample of the treated biofilm is inoculated in LB and incubated overnight. The DNA-extraction is performed on 3ml of this overnight culture with OD590 = 2. P. aeruginosa PAO1 biofilms were grown in the synthetic cystic fibrosis sputum medium (SCFM2) for 24h. The biofilm was grown in a flat bottom 96-well plate and the inoculum was approx. 5 x 107 CFU/ml. P. aeruginosa biofilms were treated with tobramycin, furanone C-30 or a combination of both in fresh SCFM2 to obtain a final concentration of 20 µg/ml tobramycin and 100 µg/ml furanone C-30. The negative control was treated with fresh SCFM2, including the same amount of DMSO (0.25%) as for the biofilms treated with C-30. After 24 h of static incubation at 37°C, biofilms were sonicated and vortexed in order to disintegrate the biofilm aggregates. A sample from this biofilm was used to start a new cycle. For each treatment three independent lineages were established, that were each exposed for 16 cycles. A sample from the treated biofilm was regrown overnight in LB and 3 ml of a culture with OD590 = 2 was resuspended in 200 µl Tris- EDTA buffer (1mM EDTA, 10 mM Tris-HCl, pH 8.5; Gibco). 100 µl of this culture was added to 500 µl of lysis buffer (50 mM Tris-HCl pH 8, 70 mM EDTA pH 8, 1% sodium dodecyl sulfate, Sigma), containing 0.5 µg/ml pronase (Roche, Mannheim, Germany) and acid washed glass beads (Sigma). After vigorous vortexing for 10 s, the samples were incubated at 37°C during 4 min. Next, 400 µl of saturated ammonium acetate was added and the samples were vortexed for 10 s prior to centrifugation (2 min, 13000 rpm). Subsequently, 600 µl of chloroform was added and the samples were vortexed for another 10 s. The phases were separated by centrifugation (5 min, 13000 rpm) and 400 µl of the top aqueous phase was transferred to a DNAse free Eppendorf tube (Lobind tube, Eppendorf, Aarschot, Belgium) containing 1 ml of 100% ethanol. After centrifugation (5 min, 13000 rpm) the pellet was washed with 500 µl of 70% ethanol and dried in a SpeedVac (Thermo Fisher Scientific, USA) for 10 min. Finally, the extracted DNA was dissolved in 100 µl of low EDTA- Tris buffer (0.1 mM EDTA, 10 mM Tris-HCl, pH 8.5) containing 0.5 µg/ml RNAse (Qiagen, Venlo, The Netherlands) and was incubated at 37°C for 1 h. The DNA was subsequently quantified using a BioDrop µLITE (BioDrop, Cambridge, UK). NEB prep kit ENA first public 2021-03-05 ENA last update 2021-02-18 External Id SAMEA8105936 INSDC center alias Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC center name Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC first public 2021-03-05T04:11:57Z INSDC last update 2021-02-18T14:20:22Z INSDC status public Submitter Id E-MTAB-10124:L3C5 tob broker name ArrayExpress compound tobramycin dose 20 genotype wild type genotype growth condition SCFM2 individual 3 organism part whole organism passage 5 sample name E-MTAB-10124:L3C5 tob strain PAO1 ERS5793011 SAMEA8105937 208964 Pseudomonas aeruginosa PAO1 Protocols: The biofilm is treated for 24h with tobramycin and C-30 alone or a combination of both for 24h. A sample of the treated biofilm is inoculated in LB and incubated overnight. The DNA-extraction is performed on 3ml of this overnight culture with OD590 = 2. P. aeruginosa PAO1 biofilms were grown in the synthetic cystic fibrosis sputum medium (SCFM2) for 24h. The biofilm was grown in a flat bottom 96-well plate and the inoculum was approx. 5 x 107 CFU/ml. P. aeruginosa biofilms were treated with tobramycin, furanone C-30 or a combination of both in fresh SCFM2 to obtain a final concentration of 20 µg/ml tobramycin and 100 µg/ml furanone C-30. The negative control was treated with fresh SCFM2, including the same amount of DMSO (0.25%) as for the biofilms treated with C-30. After 24 h of static incubation at 37°C, biofilms were sonicated and vortexed in order to disintegrate the biofilm aggregates. A sample from this biofilm was used to start a new cycle. For each treatment three independent lineages were established, that were each exposed for 16 cycles. A sample from the treated biofilm was regrown overnight in LB and 3 ml of a culture with OD590 = 2 was resuspended in 200 µl Tris- EDTA buffer (1mM EDTA, 10 mM Tris-HCl, pH 8.5; Gibco). 100 µl of this culture was added to 500 µl of lysis buffer (50 mM Tris-HCl pH 8, 70 mM EDTA pH 8, 1% sodium dodecyl sulfate, Sigma), containing 0.5 µg/ml pronase (Roche, Mannheim, Germany) and acid washed glass beads (Sigma). After vigorous vortexing for 10 s, the samples were incubated at 37°C during 4 min. Next, 400 µl of saturated ammonium acetate was added and the samples were vortexed for 10 s prior to centrifugation (2 min, 13000 rpm). Subsequently, 600 µl of chloroform was added and the samples were vortexed for another 10 s. The phases were separated by centrifugation (5 min, 13000 rpm) and 400 µl of the top aqueous phase was transferred to a DNAse free Eppendorf tube (Lobind tube, Eppendorf, Aarschot, Belgium) containing 1 ml of 100% ethanol. After centrifugation (5 min, 13000 rpm) the pellet was washed with 500 µl of 70% ethanol and dried in a SpeedVac (Thermo Fisher Scientific, USA) for 10 min. Finally, the extracted DNA was dissolved in 100 µl of low EDTA- Tris buffer (0.1 mM EDTA, 10 mM Tris-HCl, pH 8.5) containing 0.5 µg/ml RNAse (Qiagen, Venlo, The Netherlands) and was incubated at 37°C for 1 h. The DNA was subsequently quantified using a BioDrop µLITE (BioDrop, Cambridge, UK). NEB prep kit ENA first public 2021-03-05 ENA last update 2021-02-18 External Id SAMEA8105937 INSDC center alias Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC center name Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC first public 2021-03-05T04:11:57Z INSDC last update 2021-02-18T14:20:22Z INSDC status public Submitter Id E-MTAB-10124:L3C5 tob + C30 broker name ArrayExpress compound tobramycin; 20 microgram per mililiter and DMSO; 0.25 percent genotype wild type genotype growth condition SCFM2 individual 3 organism part whole organism passage 5 sample name E-MTAB-10124:L3C5 tob + C30 stimulus furanone C-30; 100 microgram per mililiter strain PAO1 ERS5793012 SAMEA8105938 208964 Pseudomonas aeruginosa PAO1 Protocols: The biofilm is treated for 24h with tobramycin and C-30 alone or a combination of both for 24h. A sample of the treated biofilm is inoculated in LB and incubated overnight. The DNA-extraction is performed on 3ml of this overnight culture with OD590 = 2. P. aeruginosa PAO1 biofilms were grown in the synthetic cystic fibrosis sputum medium (SCFM2) for 24h. The biofilm was grown in a flat bottom 96-well plate and the inoculum was approx. 5 x 107 CFU/ml. P. aeruginosa biofilms were treated with tobramycin, furanone C-30 or a combination of both in fresh SCFM2 to obtain a final concentration of 20 µg/ml tobramycin and 100 µg/ml furanone C-30. The negative control was treated with fresh SCFM2, including the same amount of DMSO (0.25%) as for the biofilms treated with C-30. After 24 h of static incubation at 37°C, biofilms were sonicated and vortexed in order to disintegrate the biofilm aggregates. A sample from this biofilm was used to start a new cycle. For each treatment three independent lineages were established, that were each exposed for 16 cycles. A sample from the treated biofilm was regrown overnight in LB and 3 ml of a culture with OD590 = 2 was resuspended in 200 µl Tris- EDTA buffer (1mM EDTA, 10 mM Tris-HCl, pH 8.5; Gibco). 100 µl of this culture was added to 500 µl of lysis buffer (50 mM Tris-HCl pH 8, 70 mM EDTA pH 8, 1% sodium dodecyl sulfate, Sigma), containing 0.5 µg/ml pronase (Roche, Mannheim, Germany) and acid washed glass beads (Sigma). After vigorous vortexing for 10 s, the samples were incubated at 37°C during 4 min. Next, 400 µl of saturated ammonium acetate was added and the samples were vortexed for 10 s prior to centrifugation (2 min, 13000 rpm). Subsequently, 600 µl of chloroform was added and the samples were vortexed for another 10 s. The phases were separated by centrifugation (5 min, 13000 rpm) and 400 µl of the top aqueous phase was transferred to a DNAse free Eppendorf tube (Lobind tube, Eppendorf, Aarschot, Belgium) containing 1 ml of 100% ethanol. After centrifugation (5 min, 13000 rpm) the pellet was washed with 500 µl of 70% ethanol and dried in a SpeedVac (Thermo Fisher Scientific, USA) for 10 min. Finally, the extracted DNA was dissolved in 100 µl of low EDTA- Tris buffer (0.1 mM EDTA, 10 mM Tris-HCl, pH 8.5) containing 0.5 µg/ml RNAse (Qiagen, Venlo, The Netherlands) and was incubated at 37°C for 1 h. The DNA was subsequently quantified using a BioDrop µLITE (BioDrop, Cambridge, UK). NEB prep kit ENA first public 2021-03-05 ENA last update 2021-02-18 External Id SAMEA8105938 INSDC center alias Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC center name Laboratory of Pharmaceutical Microbiology (LPM), Ghent University INSDC first public 2021-03-05T04:11:57Z INSDC last update 2021-02-18T14:20:22Z INSDC status public Submitter Id E-MTAB-10124:WT PAO1 broker name ArrayExpress genotype wild type genotype growth condition SCFM2 individual parent organism part whole organism passage 0 sample name E-MTAB-10124:WT PAO1 strain PAO1