RNA was extracted from day 11 colony biofilms or planktonic cultures using the RNeasy kit (Qiagen). RNA was DNase treated using Turbo DNase I (Invitrogen), and reverse transcribed using SuperScript III Reverse Transcriptase (Invitrogen). cDNA was amplified in an Applied Biosystems StepOne Plus Real-Time PCR machine using SYBR green and primers listed in Table S3. All reactions were performed using cDNA from at least three biological replicates. Each reaction was performed in triplicate with at least two different cDNA concentrations. A melt curve analysis was performed using genomic DNA and for every reaction with cDNA to verify primer specificity. Relative fold difference in transcript abundance was determined using the ΔΔCT method60. Transcripts were normalized to gyrB abundance.

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