title: 28S amplicon metagenomics of soil fungi - rainfall gradient
design: DNA was extracted from two ~0.25g aliquots of frozen soil for each sample using MoBio Power Soil Kits. DNA was quantified fluorimetrically and standardized to 10 ng uL-1. For PCR, we used Illumina TruSeq V3 indices linked to 28S rRNA fungal-specific primers NL1-NL4. For PCR reactions, we used platinum PCR Supermix (Invitrogen, Carlsbad, CA), 1.25 l of each primer (10 M), 0.5 L of BSA (20 mg mL-1), and 2 L (~20 ng) of DNA with a hot start at 93 C for 5 min, 35 cycles of 93 C for 60 s, 58 C for 60 s, 72 C for 60 s, and a final extension step of 72 C for 10 min. For each sample, PCR reactions were run in duplicate, combined, cleaned with Agencourt AMPure XP magnetic beads, and quantitated. Samples were then pooled in equal amounts and sequenced on Illumina MiSeq v3 (2 x 300 bp) at the University of Texas Genome Sequencing and Analysis Facility.