Fungal ITS1 amplicon sequencing of SF1_soil_t0_SN1
Soil DNA (Qiagen DNeasy PowerSoil) standardized to 10 ng/uL. Two-step library prep following Lumibao et al. (2018). PCR 1 amplified the fungal ITS1 region in triplicate at three annealing temperatures (52/54/56 C) with Phusion Flash HF Master Mix and adapter-tailed primers ITS1F (CTTGGTCATTTAGAGGAAGTAA) and ITS2 (GCTGCGTTCTTCATCGATGC); triplicates pooled. PCR 2 added dual indices designed after Kozich et al. (2013). Libraries purified (QIAquick), quantified on a Qubit 3.0, and pooled at 25 ng each. Sequenced at the Duke Genome Sequencing and Analysis Core Facility (Durham, NC, USA) on an Illumina MiSeq, v3 kit, paired-end 300 bp.