title: Amplicon sequencing of environmental fungi in extremes of soil moisture conditions
design: We extracted DNA from 0.25g of frozen tissue from each site at each time point using the Qiagen DNeasy PowerSoil kit (Qiagen, Germantown, MD, USA). DNA was quantified fluorometrically (Qubit, Invitrogen, Carlsbad, CA, USA) and normalized to 20 ng/l for subsequent PCR.  For PCR, we used Illumina TruSeq V3 indices (Illumina, San Diego, CA, USA) linked to ITS2 rDNA fungal-specific primers (5.8S-Fun/ ITS4-Fun; Taylor et al. 2016). These primers allow for the detection of the fungal community. Reactions contained 21.5 l of platinum PCR Supermix (Invitrogen, Carlsbad, CA, USA), 1.25 l of each primer (10M), 1.25 l of BSA (20mg/mL), and 2 l of DNA. All PCRs were performed in triplicate with a hot start at 96 C for 2 min, and 35 cycles of 94 C for 30s, 58 C for 40s, 72 C for 2 min, and a final extension step of 72 C for 10 min. Triplicate PCRs were combined and cleaned with Agencourt AMPure XP magnetic beads (Beckman Coulter, Brea, CA) and quantitated with a Qubit fluorometer. Samples were pooled in equal amounts and sequenced on Illumina MiSeq v3 (2 x 250bp PE run) at the University of Tennessee Genomics Core Facility.