title: 16S of soil
design: Sample libraries were prepared and sequenced at the Environmental Sample Preparation and Sequencing Facility at Argonne National Laboratory (Lemont, IL). For prokaryotes (archaea and bacteria), 16S rRNA genes were amplified in polymerase chain reactions (PCRs) using primers (515F/806R) that target the V4 region of the 16S rRNA gene (Parada et al. 2016); for fungi, ITS2 regions were amplified in PCR reactions using ITS9f/ITS4R primers (Ihrmark et al. 2012; Smith & Peay 2014). Each 25 L PCR reaction contained 9.5 L of MoBio PCR Water (Certified DNA-Free), 12.5 L of QuantaBios AccuStart II PCR ToughMix (2x concentration, 1x final), 1 L Golay barcode tagged forward primer (5 M concentration, 200 pM final), 1 L reverse primer (5 M concentration, 200 pM final), and 1 L of template DNA. The conditions for PCR are as follows: 94 C for 3 minutes to denature the DNA, with 35 cycles at 94 C for 45 s, 50 C for 60 s, and 72 C for 90 s; with a final extension of 10 min at 72 C to ensure complete amplification. Amplicons were then quantified using PicoGreen (Invitrogen, Carlsbad, CA) and a plate reader (Infinite 200 PRO, Tecan Group Ltd., Mnnedorf, Switzerland). Once quantified, volumes of each of the products were pooled into a single tube in equimolar amounts. This pool was then cleaned using AMPure XP Beads (Beckman Coulter) and quantified using a fluorometer (Qubit, Invitrogen, Carlsbad, CA). After quantification, the molarity of the pool was determined, diluted down to 2 nM, denatured, and then diluted to a final concentration of 6.75 pM with a 10% PhiX spike for sequencing on the Illumina MiSeq platform (Illumina Inc., San Diego, CA), resulting in 251 bp paired-end reads.