Dnaeasy powersoil pro kit

Genomic DNA was extracted from 96 pooled ECM tip samples using the DNAeasy^® PowerSoil^® Pro kit (QIAGEN, Hilden, Germany), following the manufacturer’s recommendations. First, previously dried roots were incubated for 30 min in the presence of a buffer and macerated at least three times with a sterile pestil. Next, samples were ground to a fine powder by placing the tissues in a 2.0-mL screw-cap tube containing a single 3.0-mm and five to ten 1.5-mm stainless steel beads and shaking them in a BeadBug^™ microtube homogenizer (Sigma-Aldrich, Missouri, USA) for 120 seconds at a speed of 400 rpm. The extracted genomic DNA was stored at −20°C for later use.
To amplify the internal transcribed spacers (ITS region including the 5.8S), we used the primers ITS1F [26 (link)] and ITS4 [27 ]. The PCR profiles and temperature programs were the same as in Schön et al. [28 (link)].

The frozen stool samples were thawed on ice, and DNA was extracted from them using a DNAeasy PowerSoil Pro Kit (Qiagen, Germany) according to the manufacturer's instructions with the following protocol adjustments. The liquid phase of stabilised stool samples (excess of RNAlater Stabilisation Solution) was separated by centrifugation at 10,000g for 3 min and thoroughly discarded to remove high salt content that may interfere with a subsequent DNA purification step. Next, the stabilised stool and fresh BL samples (250 mg) were bead-beaten in PowerBead Pro tubes containing proprietary beads using a Mixer Mill MM400 (Retsch, Germany) for 10, 15 or 20 min at 25 Hz. Each sample was injected with 5 µL RNase (10 mg/mL concentration; A&A Biotechnology, Poland) and incubated at 60 °C for 10 min to allow RNA digestion. This step removes RNA allowing to increase DNA yield. The DNA quality was verified with agarose gel electrophoresis. The final DNA concentration was measured by a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific, USA) (Supplementary Table 4). All the differences in the extracted DNA amount may be due to the nonhomogeneous nature of the stool sample material.