Rneasy powerplant kit

RNA was extracted from cells harvested during the feeding phase of exponentially fed cultivations. Four samples were collected at different times denoted t1–t4, where t1 is the time at which feeding was started, and t4 the time when feeding was stopped (t1 + 28 h). The control cultivations (non-adapting) were fed with a solution containing 100 g L⁻¹ glucose and 14 g L⁻¹ xylose, whereas the adapted cultivations were fed with a 40% (w/w) WSH solution, supplemented with glucose to obtain the same concentrations of fermentable sugars in both feed solutions. Samples for RNA sequencing were directly deposited in pre-cooled conical tubes, which were kept in a 50% ethanol–ice solution during sample preparation. Cells were washed using a 9 g L⁻¹ sodium chloride solution that was cooled to 4 °C and kept on ice. Cells were centrifuged at 0 °C at 3800 × g. After washing, the cell pellet was resuspended in RNAlater (Invitrogen, USA) and kept at -20 °C until analyzed. RNA was extracted using a TRIzol–chloroform method, as described by Geijer et al. [85 (link)], followed by on-column DNase digestion using the RNeasy PowerPlant Kit and RNase-free DNAse set from Qiagen (Germany). The RNA samples were analyzed with a Fragment Analyzer (Agilent, USA) and all samples were confirmed to have an RNA integrity number above 5.5.

After 1 week, when a significant increase in co-incubated N₂ fixation rates was verified (approximately 3 times higher), the cotton stoppers were replaced with the original plastic caps, and the samples were flash-frozen by immersion into liquid N₂. The tubes were kept at − 80 °C until they were freeze-dried at − 55 °C for 24 h. The dried samples were moved to RNeasy PowerBead Tubes (Qiagen, Venlo, Netherlands), which had their ceramic beads replaced with 150–212-μm acid-washed glass beads (Sigma-Aldrich, Saint Louis, USA). RNA was isolated using the RNeasy PowerPlant Kit (Qiagen) using the protocol provided by the manufacturer. DNA co-isolated from the samples was removed with the RNase-Free DNase Set kit (Qiagen). RNA in the samples was quantified with the Qubit 2.0 fluorometer (Life Technologies, Carlsbad, USA) and RNA integrity was checked with Agilent TapeStation 4200 (Agilent Technologies).