title: DNA-seq of Bishop pine forest soil
design: First step PCR amplifications were performed by mixing 1 l of template DNA, 5 l of MyTaqTM HS Red Mix, 3.2 l of PCR-grade H2O, and 0.4 l of each primer. The 515f (GTGYCAGCMGCCGCGGTAA) / 806R (GGACTACNVGGGTWTCTAAT) primer pair was used to amplify the V4 hypervariable region of the bacterial 16S rRNA gene (Caporaso et al., 2011), and the KYO1 (CTHGGTCATTTAGAGGAASTAA) / KYO2 (TAGAGGAAGTAAAAGTCGTAA) primer pair was used to amplify the ITS1 subregion of the fungal rRNA operon (Toju et al., 2012). Extraction controls and positive controls (using mock bacterial and fungal communities) were also used as DNA templates for PCR amplifications. The PCR amplifications occurred in 96-well plates and consisted of 35 cycles with a denaturation temperature of 95C, an annealing temperature of 50C (52.5C for fungi), and an extension temperature of 72C. Indexed tags for Illumina sequencing were attached during a second step PCR amplification (Toju et al., 2016), which consisted of eight cycles with identical temperature settings as the first step PCR. Gel electrophoresis was used to confirm PCR products, and a magnetic bead purification method using Sera-Mag SpeedBeads (MilliporeSigma, Munich, Germany) was used to clean PCR products. A Qubit 3.0 fluorometer (Thermo Fisher, Waltham, MA, USA) was used to quantify cleaned DNA. Lastly, we pooled our samples into two libraries at equimolar concentrations and submitted them for 2 x 300 Illumina MiSeq sequencing at the Stanford Functional Genomics Facility