16S of soil metagenome
DNA was extracted using MoBios Power Soil kit following the manufacturers instructions (MO BIO, Carlsbad, CA) and DNA quantity and quality were quantified colorimetrically via Take3 software on the Bio-Tek Synergy HT microplate reader (BioTek, Inc., Winooski, VT). Extracted DNA was diluted 10x and stored at -20C until further use. Indexed primers for the fungal ITS2 (Internal Transcribed Spacer) region and V4 region of the bacterial 16S rRNA gene were coupled to an Illumina adaptor sequence. The forward primer 515f (5-GTGYCAGCMGCCGCGGTAA-3) (Parada et al., 2016) and reverse primer 806R (5-GGACTACNVGGGTWTCTAAT-3) (Apprill et al., 2015) were used for amplification of bacteria, and the forward primer fITS7 (5-GTGARTCATCGAATCTTTG-3) (Ihrmark et al., 2012) and reverse primer ITS4 (5-CCTCCGCTTATTGATATGC-3) (White et al., 1990) were used for fungi. Triplicate PCR was performed according to (Custer et al., 2020) using Phusion High Fidelity polymerase, and correct amplification was checked on a 1.5% agarose gel. Triplicate PCR products were pooled and cleaned with Axygens AxyPrep Mag PCR Clean-up Kit by manufacturers instructions (Axygen Biosciences, Union City, CA). Concentrations of cleaned products were measured with Qubit 3.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA). An equimolar mix was prepared of the cleaned bacterial and fungal products separately for submission to the University of Minnesota Genomics Center, where sequencing was performed on the Illumina Miseq platform with version 2 chemistry resulting in paired-end reads of 250 bp length.