Comparative transcriptomics of Anabaena sp. PCC7120 grown as biofilm under bubbling or their concurrent planktonic fractions
Comparative transcriptomics of Anabaena sp. PCC7120 grown as biofilm under bubbling or their concurrent planktonic fractions
Anabaena sp. PCC7120 was grown in bespoke bubbling flasks from 0.25 OD750nm in 350 mL of fresh BG11, under 15 µmol photons m-2 s-2 -for 72 hours. Sessile (biofilm) and planktonic fractions were collected separately and their supernatants removed by centrifugation. Pooled cultures of either type were distributed into 80-100 mg wet mass samples. Cell pellets were resuspended in 600 µl of 50 mM Tris-HCl pH 8, 100 mM EDTA and 130 µl of chloroform and mixed thoroughly by inversion. The suspension was centrifuged at 12000 x g for 5 min, supernatants were removed and obtained pellets were flash frozen in liquid nitrogen for storage at -80ªC. Frozen pellets were suspended in 300 µl of a buffer containing 0.3 M sucrose and 10 mM sodium acetate at pH 4.5, to which 100 µl of 250 mM Na2-EDTA (pH 8.0), 400 µl lysis buffer (2% SDS, 10 mM sodium acetate, pH 4.5) and 1 ml of acid phenol at 65 °C were added. Samples underwent three cycles of 30 second vortexing and 2.5 min incubation at 65ºC before centrifugation at 12000 x g for 5 min. The aqueous phase was collected and RNA extracted with Trizol, Trizol-Chloroform and Chloroform, thoroughly mixing with each step. The final aqueous phase was mixed with 2 volumes of chilled absolute ethanol and precipitated O/N at -80ºC. After 30 min of centrifugation at 12000 x g, pellets were washed with chilled 70% ethanol, resuspended in nuclease-free water (Ambion) and treated with RNAse-free DNAse I (Roche). DNA elimination was ensured by Real time RT-PCR using housekeeping gene rnpB (Vioque et al., 1992). RNA was quantified spectrophotometrically using a SPECORD® PLUS Analytik Jena spectrophotometer. NGS sequencing project started with quality control of the total RNA, where the RNA integrity number (RIN) and concentration are analysed to ensure it is of sufficient integrity and quantity. Library construction was performed by STAB VIDA Lda (Lisbon, Portugal), employing a Ribosomal Depletion Library Preparation Kit for library construction.