16S amplicon metagenomics of soils : rainfall experiment
DNA was extracted from two ~0.25g aliquots of frozen soil for each sample using MoBio Power Soil Kits. DNA was quantified fluorimetrically and standardized to 10 ng uL-1. For PCR, we used Illumina TruSeq V3 indices linked to 16 rRNA bacteria-specific primers S-D-Bact-0341-b-S-17 and S-D-Bact-0785-a-A-21. For PCR reactions, we used platinum PCR Supermix (Invitrogen, Carlsbad, CA), 1.25 l of each primer (10 M), 0.5 L of BSA (20 mg mL-1), and 2 L (~20 ng) of DNA with a hot start at 95 C for 5 min, 25 cycles of 95 C for 40 s, 55 C for 2 min, 72 C for 60 s, and a final extension step of 72 C for 7 min. For each sample, PCR reactions were run in duplicate, combined, cleaned with Agencourt AMPure XP magnetic beads, and quantitated. Samples were then pooled in equal amounts and sequenced on Illumina MiSeq v3 (2 x 250 bp for bacteria) at the University of Texas Genome Sequencing and Analysis Facility.