Aerobic_biofilm_day150_3_16S
The purity and integrity of extracted DNA samples were verified using agarose gel electrophoresis (AGE). Bacterial 16S rRNA genes were PCR-amplified with forward primer 338F (5-ACTCCTACGGGAGGCAGCA-3) and reverse primer 806R (5-GGACTACHVGGGTWTCTAAT-3) targeting the V3 and V4 regions [41]. The PCR reaction mixture was 20 L, including 4 L 5  FastPfu buffer, 2L dNTPs (2.5 M), 0.8 L of forward primer (5 M), 0.8 L of reverse primer (5 M), 0.4 L FastPfu Polymerase, and 10 ng of template DNA. The PCR thermal program was as follows: an initial denaturation at 95 C for 3 min, followed by 27 cycles of denaturation at 95 C for 30 s, annealing at 55 C for 30 s, extension at 72 C for 45 s, and a final extension at 72 C for 10 min. Sequencing of amplicons from all the samples was conducted on the Illumina Miseq PE300 platform by Majorbio Biopharm Biotechnology Co., Ltd. (Shanghai, China).