title: ITS (ITS1f/ITS2 region) metagenome of leaf tissue from Vitis labrusca x Vitis riparia cv. Alpha: Alpha (Sick) plant
design: 50 mg of V. amurensis tissue was taken 800 l buffer with an 2*content of salt (100 mM Tris pH 7.5, 1.4 M NaCl, 40 mM EDTA pH 7.5, 1% CTAB) was added, incubated for 1 hour 60C in a water bath WB-4MS (BioSan, Latvia). Then 300 l of chloroform was added, gently mixed and centrifuged for 10 min 4 13,200 rpm (Ependorf, USA). Further, a supernatant was selected in separate test tubes with 950 l of 96% ethanol and frozen at -20C overnight. Next, the DNA in ethanol was centrifuged 10 min 4 13,200 rpm, after which the supernatant was removed and the precipitate was dried until the ethanol completely evaporated at room temperature. The precipitate was dissolved in 100 l of distilled water and then purified on Plasmid DNA purification columns Plasmid MiniPrep kit per manufacturers protocol (Evrogen, Moscow, Russia). As a result, the DNA was dissolved in 50 l of water. DNA was assessed for quality and quantity using the Nanodrop-1000 (Nanodrop, USA) and Quantus Fluorometer (Promega, USA), respectively. DNA samples were sent to Syntol (Moscow, Russia) for high-throughput sequencing using Illumina technology. Libraries were prepared for sequencing according to the protocol described in the manual "16S Metagenomic Sequencing Library Preparation" (Part # 15044223 Rev. B; Illumina). Fungal ITS1 rDNA regions were amplified from all samples using primers ITS1f (5CTTGGTCATTTAGAGGAAGTAA) and ITS2 (5 GCTGCGTTCTTCATCGATGC). The library pool was sequenced on Illumina MiSeq (2 x 250 paired end) using MiSeq Reagent Kit v2 (500 cycles).