title: Metagenomic sequencing of Missouri maize soils: fungi Bradford additional baseline depth in time 0 rep 3 pre nitrogen
design: Soil microbial DNA was extracted using the MoBio PowerSoil DNA Isolation Kit with slight modifications as recommended by Lindahl et al. (2013). Samples were homogenized using a Vortex-Genie 2 Mixer for 10 to 15 minutes. The DNA templates were quantified using an ND1000 spectrophotometer and then concentrated to a final concentration of 30 ng l-1.  For the generation of the amplicon library, fungal molecular identification was conducted using the nuclear internal transcribed spacer 1 (ITS1) region. A two-step PCR approach was employed, where the initial PCR amplification involved the use of forward primer ITS1F_KYO1 and reverse primer ITS2_KYO2. The PCR conditions included an initial denaturation at 94C for 2 min, followed by 35 cycles of denaturation at 98C for 10 sec, annealing at 50C for 30 sec, extension at 68C for 50 sec, and a final extension at 68C for 5 min. A final PCR step was performed to ligate Illumina adapters and barcodes. The amplicon libraries were then sequenced at the Brigham Young University DNA sequencing center using the Illumina HiSeq 2500 platform with paired-end sequencing (2x250).