Three discs were removed from the model every week and the biofilms on these discs were dispersed in 1mL sterile water by sonicating the discs for 1 min, pelleted for 15 min at 4500 rpm and stored at −80 °C for sequencing. The DNA of all samples was extracted and purified according to Cieplik et al. (Cieplik et al., 2019 (link)). In brief, the samples were added to wells of a 96-deep-well plate containing Tris-saturated phenol, 0.1 mm zirconium beads and lysis buffer and were mechanically lysed by bead-beating at 1,200 rpm for 2 min. DNA was isolated with the Mag MiniKit (LGC Genomics, Berlin, Germany).
Quantitative PCR was used to determine the bacterial DNA concentration in the biofilm samples, using universal primers specific to the bacterial 16S rRNA gene (Ciric et al., 2010 (link)). The V4 hypervariable region of the 16S rRNA gene was amplified using 1 ng DNA with 1 µM of each primer and 30 amplification cycles (Caporaso et al., 2011 (link)). Paired-end sequencing of the DNA was conducted on the MiSeq platform (Illumina, San Diego, CA, USA) with a MiSeq Reagent kit v3 and 2x251 nt at the VUmc Cancer Center Amsterdam (Amsterdam, the Netherlands). The sequence and meta data are available in the NCBI BioProject database under accession numberPRJNA614901 .
Quantitative PCR was used to determine the bacterial DNA concentration in the biofilm samples, using universal primers specific to the bacterial 16S rRNA gene (Ciric et al., 2010 (link)). The V4 hypervariable region of the 16S rRNA gene was amplified using 1 ng DNA with 1 µM of each primer and 30 amplification cycles (Caporaso et al., 2011 (link)). Paired-end sequencing of the DNA was conducted on the MiSeq platform (Illumina, San Diego, CA, USA) with a MiSeq Reagent kit v3 and 2x251 nt at the VUmc Cancer Center Amsterdam (Amsterdam, the Netherlands). The sequence and meta data are available in the NCBI BioProject database under accession number
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