title: Microbiome of untreated soybean soil
design: Total genomic DNA was extracted from 0.25g of soil using the PowerSoil DNA Isolation Kit (Qiagen, USA) following the manufacturers instructions. The extracted DNA samples were analyzed for quality and quantity using NanoDrop 1000 (Thermo Scientific, Wilmington, DE, USA) and were verified with agarose gel electrophoresis. A  2 x 250 bp paired-end sequencing following the 16S Illumina Amplicon Protocol from the Earth Microbiome Project (https://earthmicrobiome.org/protocols-and-standards/16s/). The standard library preparation protocol was conducted by amplifying the  V4 region of the 16S small subunit rRNA gene  using the universal primer set, 515F (GTGYCAGCMGCCGCGGTAA) and 806R (GGACTACNVGGGTWTCTAAT).  The PCR reaction mixture included 13.0 L of PCR-grade water, 1.0 L of template DNA, 10 M of each primer, and 10.0 L of 5PRIME HotMasterMix (2x) (Quantabio, Beverly, MA). Samples were amplified in duplicate under the following thermocycler conditions: 94C for 3 min for initial denaturing, then 35 cycles of 94C for 45 s, 50C for 60 s, and 72C for 90 s. A final elongation step occurred at 72C for 10 min followed by a hold at 4C. After pooling duplicates, all amplicons were visualized on a 2% agarose gel and quantitated on a Qubit fluorometer (FisherScientific, Hampton, NH). Normalization was performed based on Qubit results and amplicons were pooled. The pool was quantitated by qubit and tapestation to determine size (380bp). The pool was then quantitated using qPCR