GSM5856278: P. aeruginosa_single biofilm [PA3-N0788]; Pseudomonas aeruginosa; RNA-Seq
Total RNA was extracted using RNeasy mini kit (QIAGEN) and as described in França et al., 2012, PLoS One 7 (5): e37480. In brief, cells were suspended in 600 µL of the lysis buffer provided by the kit, plus 500 µL of phenol and 12 µL of β-mercaptoethanol. This suspension was transferred to a safe lock tube (2 mL) with 0.4 g of acid-washed 150–212 mm glass beads (Sigma) and, using a BeadBugTM 6 (Benchmark Scientific) cells disruptor, the cells were lysed (4× 4500 rpm for 35 seconds, with 5 minutes intervals on ice between cycles). Finally, the tubes were centrifuged, and the suspension was transferred into a new tube. An equal volume of 70% of ethanol was added, the suspensions transferred into the RNeasy mini kit columns and the manufacturer's instructions were strictly followed. RNA quality was determined using Agilent TapeStation 4200 (Agilent) and RNA quality indicators were above 8 for all samples. Thereafter, total RNA obtained from three independent experiments were mixed and treated with TURBO DNase (Ambion) to degrade genomic DNA. Additionally, before the library construction, a NEBNext rRNA Depletion Kit (Bacteria) (NEB) was used to remove bacterial ribosomal RNA. RNA libraries were prepared by strictly following the instructions of the kit KAPA HyperPrep (Roche). Libraries quality was determined using Agilent TapeStation 4200 and data were generated using Illumina NovaSeq 6000 (Illumina) from paired-end read (2 × 150 bp).