Soil bacterial community response to cover crops, cover crop termination, and predicted climate conditions
The V3-V4 region of the 16S rRNA gene was amplified through 25 cycles of PCR with barcoded and indexed primers using the Q5 High-Fidelity Mastermix (New England BioLabs, Inc.) with 25  of mix, 6_ molecular-grade water, 21_ of forward and reverse barcoded primer mix at 1.09 uM concentration, and 4_ sample DNA. The PCR protocol was as follows: 98_ for 30 seconds; 30 cycles of denaturation at 98_ for 10 seconds, annealing at 55_ for 30 seconds, elongation at 72_ for 30 seconds; then a final elongation at 72_ for 2 minutes. Primers included the MiSeq adaptors (A for forward, B for reverse), the sample index/barcodes, the two-nucleotide linker, and primers 341F (5_-ACTCCTACGGGAGGCAGCAG-3_) (Fadrosh et al., 2014) and 806R (5_-GGACTACHVGGGTWTCTAAT-3_) (Caporaso et al., 2011). Amplicons were purified using Omega Mag-Bind beads (Omega Bio-Tek, Norcross, Georgia) and quantified with a Qubit 4 Fluorometer (Invitrogen, Thermofisher Scientific). High-throughput sequencing was performed using a 2 x 300-nucleotide V3 kit and Illumina MiSeq (Illumina, San Diego, CA) at the University of Oregon Genomics Core (Eugene, OR, USA). PhiX at a 10% spike-in and molecular-grade sterilized water were used as positive and negative controls respectively.