ITS sequencing of bulk soil: soil with rye and hairy vetch mixed cover crops replicate 1 in 2018 year of experiment
The first amplification was done using the primers ITS1 and ITS2 in PCR conditions: 94 for 4 min; 94 for 1 min / 55 for 1 min / 72 for 2 min (31 cycles); 72 for 10 min. PCR reaction (total 20 l): 10 L AmpliTaq Gold 360 Master Mix (AB Applied Biosystems, USA), 0.8 L of each primer, 13 L of template DNA, and 5.47.4 L of nuclease-free water. The product was purified using AMPure XP reagent (Nipon Genetics Co. Ltd., Japan). The second amplification was done to the purified first PCR product in 20 l of total PCR reaction using the same primers attached with barcodes and adaptor in PCR condition: 94 for 4 min; 94 for 1 min / 55 for 1 min / 72 for 2 min (10 cycles); 72 for 10 min. Products are pooled equimolar to a final concentration of 50 pM and sequenced using the Ion PGM Hi-Q Chef kit.