title: Amplicon sequencing of environmental bacteria in extremes of soil moisture conditions
design: We extracted DNA from 0.25g of frozen soil from three soils types at different time points in the incubation using the Qiagen DNeasy PowerSoil kit (Qiagen, Germantown, MD, USA). DNA was quantified fluorometrically (Qubit, Invitrogen, Carlsbad, CA, USA) and normalized to 20 ng/l for subsequent PCR.  For PCR, we used Illumina TruSeq V3 indices (Illumina, San Diego, CA, USA) linked to 16S rRNA bacterial-specific primers (341F/785R; Klindworth et al., 2013). These primers allow for the detection of the bacterial community. Reactions contained 21.5 l of platinum PCR Supermix (Invitrogen, Carlsbad, CA, USA), 1.25 l of each primer (10M), 1.25 l of BSA (20mg/mL), and 2 l of DNA. All PCRs were performed in triplicate with a hot start at 95 C for 5 min, and 25 cycles of 95 C for 60s, 55 C for 2 min, 72 C for 60 s, and a final extension step of 72 C for 7 min. Triplicate PCRs were combined and cleaned with Agencourt AMPure XP magnetic beads (Beckman Coulter, Brea, CA) and quantitated with a Qubit fluorometer. Samples were pooled in equal amounts and sequenced on Illumina MiSeq next gen sequencing (2 x 300bp PE run) at the University of Tennessee Genomics Core Facility.