Metabarcoding of fungal ITS2: Grassland_Amtvik_high_catenary_position_plot8_soil_10-20cm
Total DNA was extracted from 100 mg (soil depth 0-30 cm) or 400 mg (soil depth 40-50 cm) freeze-dried, homogenized soil using Macherey-Nagel NucleoSpin Soil kit (Dren, Germany). Fungal community amplicons were generated by PCR amplification of ITS2 markers using the forward primer gITS7 and the reverse primer ITS4 and ITS4arch elongated with a unique 8 bp long identification tag for each sample. PCRs were run in technical duplicates and template concentrations and number of PCR cycles were optimized for each sample. Amplicons were purified using the AMPure kit (Beckman Coulter, California, USA), concentrations were measured fluorometrically (Qubit Fluorometer, Invitrogen, Carlsbad, California, USA) and products mixed in equal amounts into four sequencing pools. Adaptor ligation and Pacific Biosciences Sequel I sequencing (Pacific Biosciences, Menlo park, California, USA) were performed by SciLifeLab (National Genomics Infrastructure, Uppsala, Sweden).