16S-based bacterial assemblage of corn rhizosphere
DNA was extracted using the Powersoil DNA Isolation Kit (MoBio Laboratories, CA, USA). DNA extracts were quantified using a Qubit Fluorometer and the dsDNA HS Assay kit (Life Technologies, CA, USA). PCR primers (515F/806R) designed by Caporaso et al. (J. G. Caporaso et al., 2012) were used to amplify the bacterial V4 hypervariable region of the 16S rRNA gene.  PCR reagent mixes contained 12.5 ľl KAPA HiFi HotStart Ready Mix (2x), 0.75 ľl each of the forward and reverse primers (10 ľM final concentration), 10 ng genomic DNA, and PCR water for a total reaction volume of 25 ľl. The following touchdown PCR conditions were used:  initial denaturation at 95?C for 2 min followed by 32 cycles of denaturation at 98?C for 20 sec, annealing beginning at 61?C and ending at 50?C for 30 sec, and extension at 72?C for 30 sec. The annealing temperature was lowered 1?C every cycle until reaching 50?C, which was used for the remaining cycles. Following this, a final extension of 72?C for 10 min was used. PCR products were purified by ethanol precipitation and verified on a 1% agarose gel. Positive amplicons were quantified using a Qubit Fluorometer and the dsDNA HS Assay kit (Life Technologies, CA, USA).   Amplicons were pooled at equimolar concentrations, and the resulting pooled library was size-selected to remove smaller primer dimers. Since the 16S rRNA gene amplicon was approx. 420 bp, the E.Z.N.A. NGS Clean-IT Kit (Omega Bio-Tek, GA, USA) was used on the pooled bacterial 16S rRNA gene libraries, removing <150bp fragments. Libraries were quantified using a Qubit Fluorometer and dsDNA HS Assay kit (Life Technologies, CA, USA), denatured with 0.2 N NaOH, and diluted with pre-chilled HT1 buffer (Illumina, CA, USA). The denatured sample library was diluted, spiked with denatured PhiX, and sequenced on an Illumina MiSeq (Illumina, CA, USA).
SUB1186947
MC3-9