Dneasy powerclean cleanup kit

The collected samples were transported to Cell Biotech, Co., Ltd. (Gyeonggi-do, Korea) and immediately frozen at −80 °C. A total of 204 samples were used for microbiome analysis. Microbial DNA was extracted using the FastDNA SPIN Kit for Soil (MP Biochemicals, Santa Ana, CA, USA) according to the manufacturer’s instructions. The extracted microbial DNA was purified using DNeasy PowerClean Cleanup Kit (Qiagen, Hilden, Germany) and then DNA quality was measured using NanoDrop (Thermo Fisher Scientific, Carlsbad, CA, USA). The purified DNA was measured for DNA concentration using the Qubit™ dsDNA BR Assay kit (Thermo Fisher Scientific). A sequencing library was prepared according to the Illumina 16S Metagenomic Sequencing Library Preparation Guide (Illumina, San Diego, CA, USA). The V4-V5 region of the bacterial 16S rRNA gene was amplified for 16S rRNA gene sequencing. The forward primer in the v4 region (CCA GCM GCC GCG GTA ATW C) and the reverse primer in the v5 region (CC GTC AAT TYY TTT RAG TTT) were used for PCR amplification in this study. The amplified sequencing library was purified with Agencourt^® AMPure XP beads (Beckman Coulter, Brea, CA, USA) and the quality of the library was confirmed using a 2100 Bio-analyzer (Agilent, Santa Clara, CA, USA). The library pool was sequenced with 250 bp paired-end reads on the MiSeq platform using the MiSeq reagent kit V2 (Illumina).

For metabarcoding, we extracted DNA from approximately 10 g of each soil sample using Qiagen DNeasy PowerMax Soil Kit. We used all samples collected or 21 per timepoint (63 total samples). Samples from each timepoint were extracted and sequenced separately (Table 1). We purified extracted genomic DNA using Qiagen DNeasy PowerClean Cleanup Kit. We used universal metazoan primers mlCOIint and HCO2198 from Leray et al. (2013 (link)) to amplify a fragment of the COI gene. Each reaction contained 10 µl of Phusion High‐Fidelity Master Mix (Thermo Fisher Scientific), 0.4 µl of each of the forward and reverse primers, 6.7 µl water, and 2.5 µl DNA. The reactions were run with the following protocol: [98°C × 3 min, 27× (98°C × 10 s, 46°C × 30 s, 72°C × 45 s), 72°C × 5 min]. Each sample was analyzed with three replicate PCRs. After verifying the PCRs’ success on 1.5% agarose gels, we pooled the three replicates together (21 total pools per timepoint) and quantified their DNA concentration using a Qubit. Sequencing was performed on a MiSeq v2 Nano flow cell with 2 × 250 bp paired‐end reads at the Genomics Core of the Research Technology Support Facility (RTSF) at Michigan State University. Raw reads were demultiplexed by the RTSF.