16s (V4 region) metagenome of leaf tissue from Polygonum cuspidatum: Pc-1 plant
50 mg of Polygonum cuspidatum 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). Bacterial 16S rRNA regions were amplified from all samples using the modified for plants primers 515F (5GGTAATACGKAGGKKGCDAGC) and 806R (5RTGGACTACCAGGGTATCTAA). The library pool was sequenced on Illumina MiSeq (2  250 paired end) using MiSeq Reagent Kit v2 (500 cycles).