Sureclean plus solution

For RNA isolation 2 mL of reactor liquid samples were used. The samples were centrifuged at 12,000 rpm for 10 min. RNA extractions were carried out with the Zymo Research Soil/Fecal RNA kit (R2040, Zymo Research, Irvine, CA, United States). After lysis (bead beating), the Zymo Research kit protocol was followed. The DNA contamination was removed by Thermo Scientific Rapidout™ DNA removal kit (K2981, Thermo Fisher Scientific, Waltham, MA, United States). Ribosomal RNA was depleted using the Ribo-Zero™ rRNA Removal Kit for Bacteria (Illumina, Madison, USA) according to the manufacturer’s instructions. The rRNA depleted samples were purified via the RNA Clean and Concentrator Columns from Zymo Research (Irvine, USA). During this step, an additional in-column DNase I treatment was included to ensure complete removal of DNA. Subsequently, synthesis of double-stranded cDNA was conducted using the Maxima H Minus Double-Stranded cDNA Synthesis Kit from ThermoScientific (Waltham, USA). In the first-strand cDNA synthesis reaction, 2 μL of random hexamer primer were used. Final purification of the blunt-end double-stranded cDNA was carried out using SureClean Plus solution from Bioline (Luckenwalde, Germany). The cDNA was sequenced in the same way as the total DNA. The quality of the RNA preparation was checked by agarose gel electrophoresis (data not shown).

A biogas fermenter sample was taken in March 2015, mixed with an equal amount of RNAlater solution and immediately frozen on dry ice for transport. In the lab, the sample was stored at −70 °C.
Isolation of ribonucleic acids for RNA-Seq was carried out using the PowerMicrobiome™ RNA Isolation Kit from Mo Bio Laboratories (Carlsbad, Germany), as recommended by the manufacturer. In a next step, ribosomal RNA was depleted using the Ribo-Zero™ rRNA Removal Kit for Bacteria (Illumina, Madison, USA) according to the manufacturer’s instructions. The rRNA-depleted samples were purified via the RNA Clean & Concentrator Columns from Zymo Research (Irvine, USA). During this step, an additional in-column DNase I treatment was included to ensure complete removal of DNA. Subsequently, synthesis of double-stranded cDNA was conducted using the Maxima H Minus Double-Stranded cDNA Synthesis Kit from ThermoScientific (Waltham, USA). In the first-strand cDNA synthesis reaction, 2 µl of random hexamer primer were used. Final purification of the blunt-end double-stranded cDNA was carried out using SureClean Plus solution from Bioline (Luckenwalde, Germany). The cDNA was sequenced in the same way as the total DNA. To achieve the required amount of cDNA for library preparation, multiple RNA isolations from the same sample were pooled.