title: Fungal ITS1 MiSeq amplicons from soil, litter, root, and wood samples
design: Total genomic DNA was extracted using different methods across the four substrates: the litter and soil samples with PowerSoil kits (MoBio, Carlsbad, CA, USA), the wood samples with the PowerPlant Pro kit (MO-BIO laboratories, Carlsbad, CA, USA, and the root samples with a standard CTAB chloroform extraction as outlined in Kennedy et al. (2003). For the three kit-based extractions, manufacturer's instructions were followed with no modifications. The starting material for each sample was as follows: 250 mg litter, soil, and wood and 20 mg of fine root material. All samples were pre-homogenized via grinding and/or bead-beating. Negative controls for each sample type were also extracted. For the Illumina MiSeq sequencing, the ITS1 gene region was amplified using the primers and protocols of Smith and Peay (2014). Illumina PCR reagents and conditions were as follows: 1 μl template DNA, 10.75 μl nuclease-free water, 0.16 μl GoTaq (Promega, Madison, WI, USA), 2 μl 10X buffer with MgCl2, 2 μl dNTP mix (2.5 mM), 2 μl Bovine Serum Albumin (10 mg/ml), 0.4 μl forward primer (10 μM), 0.4 μl reverse primer (10 μM). PCR cycling conditions included 95°C for 2 minutes, followed by 29 cycles of 95°C for 30 seconds, 54°C for 30 seconds, 72°C for 1 minute, and then 72°C for 5 minutes. After confirming amplification via gel electrophoresis, samples were cleaned using either the Charm 'Just-A-Plate' kit (Charm Biotech, San Diego, CA, USA) for MiSeq sequencing.  Following cleaning, samples were added in equal concentration to respective libraries for sequencing at the University of Minnesota Genomic Center. The MiSeq sequencing was done using 2 x 250 bp V2 Illumina chemistry.