Sample 4
soil metagenome
Protocols: samples were taken from the silage digestate in the HR digested for 2 d. At this time point, high AD rates were detected as indicated by the fast increase of volatile fatty acids (VFA), e.g., acetic acid. Sampling was performed at two different organic loading rates (OLR), i.e., batch-fermentation of 500 g (denominated as “low OLR”, samples MOLR500 and TOLR500) and 1,500 g silage (denominated as “increased OLR”, samples MOLR1500 and TOLR1500) The total microbial RNA was isolated applying the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's guidelines. For each sample, RNA from two subsamples (replicates) were extracted. Subsequently, the RNA was purified using the Ambion® Turbo DNA-free Kit (ThermoFisher, Germany). Ribosomal RNA was depleted using the Ribo-Zero™ rRNA Removal Kit for Bacteria (Illumina, Madison, USA) according to the manufacturer's instructions. The remaining mRNA transcripts were fragmented to approximately 550 bp fragments using a focused-ultrasonicator (Covaris M220, Woburn, USA). cDNA libraries for Illumina sequencing were constructed using the TruSeq RNA Library Prep Kit v2 (Illumina, Eindhoven, Netherlands).