16s rRNA amplicon sequencing of grassland soil
Genomic DNA was extracted from soils using a MoBio PowerSoil Extraction kit according to the manufacturers instructions. Successful genomic DNA (gDNA) extraction was confirmed using a NanoDrop spectrophotometer. The bacterial and archaeal 16S rRNA gene was targeted using universal bacterial primers (515F/806R) and amplified using PCR according to Earth Microbiome Project protocols (Caporaso et al. 2012) with a few exceptions. First, we added 2 l of 1% Bovine Serum Albumin, 0.25 l of MgCl2 and double the amount of gDNA to each reaction well. Additionally, PCR was only run for 25 cycles instead of 35. Each sample was amplified in triplicate, and amplification was confirmed using gel electrophoresis. Each sample was normalized by DNA concentration and combined into a single amplicon library. The combined library was cleaned using a QIAquick Gel Extraction Kit according to included instructions. The library was sequenced using a 2 x 150 paired-end Illumina MiSeq run, with v2 reagents and a 10% PhiX spike, in the Integrated Genomics Facility at Kansas State University (Manhattan, KS, USA).