As negative control, one high volume culture was set up with a medium without being supplemented with any substrate. Cultures were incubated at 28 °C under shaking by using baffled Erlenmeyer flasks until mid-exponential phase (OD 0.6–0.9) was reached (incubator: INE 800, Memmert, Schwabach, Germany; shaker:
KS501, IKA Labortechnik, Staufen, Germany). Starting from two pre-cultures (50 mL) which had been transferred twice after having been grown to mid exponential phase on glucose, three cultures (50 mL) per substrate of interest (chondroitin sulfate, λ-carrageenan, fucoidan or glucose as reference, 1.8 g/L) were prepared with a 10% (v/v) inoculum (5 mL). The initial OD600 nm was determined and monitored over one week. As negative control, three cultures had no substrate. As positive control, three cultures were grown on medium M13a supplemented with casamino acids (Schlesner, 1994). Growth curves PD0332991 purchase allowed the calculation of growth rates and doubling times. Cell material for downstream processing was harvested by centrifugation and was kept at − 20 °C (− 80 °C for long term storage) until it was processed. Stored cell pellets were thoroughly resuspended in 1–3 mL of TRI reagent (Applied Biosystems, Darmstadt, Germany). The suspension was incubated for 5 min at room temperature. Cells were lysed by beadbeating (lysing matrix B, material: 0.1 mm silica spheres;
MPBiomedicals, Berlin, Germany) applying a FastPrep 24 automated homogenizer (MPBiomedicals). Three steps of 30 s (speed:
6 m/s) were performed, while cooling PR 171 the tubes on ice between beadbeating steps. After the third step, the beadbeater tubes were incubated on ice for additional 10 min. Next, beadbeater tubes were centrifugated at 4 °C for 10 min (5415 C, Eppendorf, Hamburg, Germany; 16,000 × g). Supernatants were transferred into RNase-free, sterile 1.5 mL Eppendorf cups. 200 μL of ice-cold chloroform was added per sample. Suspensions were thoroughly mixed by vortexing for 20 s, followed by a 10 min incubation step at RT. A further centrifugation step was carried out (4 °C, 15 min, 16,000 × g). The aqueous, upper phase was transferred into new, RNase-free and sterile Eppendorf cups. 1 mL of 100% isopropanol was added, followed Vasopressin Receptor by incubation at − 20 °C for 1 h. After the incubation, a 30 min centrifugation step was performed (4 °C, 16,000 × g). The supernatants were discarded and pellets were washed twice in 75% ethanol. Dried pellets were dissolved in 50–100 μL RNase-free water. Extracted RNA was cleaned by using the RNeasy MinElute clean-up kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. The concentration and quality of eluted RNA were determined by using a NanoDrop® spectrophotometer (Thermo Scientific, Wilmington, USA). The amount and quality of extracted and cleaned up RNA were also documented by RNA agarose gelelectrophoresis.