title: Illumina Miseq 16S B.gracilis rhizosphere
design: To obtain high-resolution representation of the soil microbial community and its response to drought, rRNA gene amplicon sequencing was performed on all replicates of bulk and rhizosphere soil for all treatment and pretreatment samples (n=60). Genomic DNA was extracted from 0.5 g frozen soil samples using the Fast DNA SPIN Kit for soil (MP Biomedicals, Santa Ana, CA) according to the manufacturers instructions. We targeted the V4 region of the 16S rRNA gene using f515a and r806b (GTGYCAGCMGCCGCGGTAA and GGACTACVSGGGTATCTAAT, respectively) from the Earth Microbiome Project (Thompson et al., 2017), and the D2 hypervariable region of the fungal LSU using LR22r and LR3 (Mueller et al., 2016). PCR reactions were carried out in 20 L volumes containing 1 U Phusion high-fidelity polymerase (ThermoFisher Scientific, MA, USA), a final concentration of 1x Phusion HF reaction buffer, 200 M dNTPs, 0.5 M each primer, 1 M BSA, 1 mM MgCl, sterile molecular grade water, and 5 L (10 ng) genomic DNA as a template. PCR conditions consisted of initial denaturation at 98 C for 30s, followed by 22 cycles of 98 C for 30 s, 55 C for 30 s, 72 C for 45 s and a final extension at 72 C for five minutes. Samples were purified using AMPure magnetic beads and barcoding was conducted using the Illumina Nextera Indexing Kit D, with 10 cycles of indexing PCR in 20 L volumes using the same concentrations as PCR1 with 10 L template DNA. Samples were purified again with magnetic beads and PCR products were checked for quality and length in a 1% agarose gel and quantitated using the Quant-iT dsDNA Kit (Invitrogen, Carlsbad, USA) with a BioTek H2 plate reader. Individual samples for each community were combined at equimolar concentrations and the two libraries were pooled at 4 nM and loaded onto an in-house Illumina MiSeq (Illumina, San Diego, USA) and sequencing using the V3 600 cycle kit.