title: 16S rRNA amplicon of rhizosphere bacterial community
design: Rhizosphere samples were thawed from -80C storage for an hour, weighed to 250 mg, and placed into a 96 well plate for DNA isolation using the DNeasy PowerSoil HTP 96 Kit (MoBio Laboratories, Inc., Carlsbad, CA, USA). At Step 30 in this protocol, the final elution volume was modified to 50 L of Solution C6, which sat on the membrane at room temperature for 5 minutes before the final centrifuge step. All isolated DNA was then transferred into sterile 1.5 mL tubes and stored at -80C until 16S PCR amplification. DNA concentrations for each sample were verified using a NanoDrop 2000 Spectrophotometer (NanoDrop Products, Wilmington, DE, USA). For bacterial communities, the V3-V4 region of the 16S rRNA gene was targeted using the primer pair 341F and 785R during the initial amplification PCR step. The sequences for 341F and 785R are 5-CCTACGGGNGGCWGCAG-3and 5-GACTACHVGGGTATCTAATCC-3 respectively (Klindworth et al. 2013; Thijs et al. 2017). 5 L at 1M of the forward and reverse primers and 12.5 L of the KAPA HiFi HotStart Ready Mix (KAPA Biosystems, Wilmington, MA, USA) were combined with 2.5 L of DNA template per reaction. PCR conditions were set for 95C for 3 minutes, followed by 25 cycles of 95C for 30 seconds, 55C for 30 seconds, and 72C for 30 seconds with a final elongation step of 72C for 5 minutes. All PCR products were then cleaned with 20 L Agencourt AMPure XP magnetic beads (Beckman Coulter, Brea, CA, USA) followed by two 200 L washes of freshly prepared 80% ethanol. Remaining ethanol was then removed, and PCR products were allowed to dry for 10 minutes before they were resuspended in 50 L of Tris-HCl. Purified PCR products were then stored at -20C until multiplexing steps. Samples were multiplexed with specifically designed index primers that allow for bridge amplification and cluster generation on the Illumina MiSeq flow cell (Illumina Corporation, San Diego, CA, USA). Each sample was multiplexed with a unique combination of forward and reverse index primers for sample identification in downstream bioinformatics and hypothesis testing. These primers were sourced from the i5/i7 Nextera XT Index Kit. This index PCR used a total volume of 50 L that consisted of 5 L of purified PCR product from the previous reactions, 5 L of forward and reverse index primers, 25 L of KAPA HiFi HotStart Ready Mix, and 10 L of sterile nuclease free water. This PCR was performed on the following conditions: an initial cycle of 95C for 3 minutes followed by an 8-cycle sequence of 95C for 3 seconds, 55C for 30 seconds, and 72C for 30 seconds with a final elongation period held at 72C for 5 minutes. Index PCR products (50 L per rhizosphere sample) were purified using 56 L of Agencourt AMPure XP magnetic beads and two washes of 220 L of freshly prepared 80% ethanol. Cleaned product was then resuspended in 25 L Tris-HCl after all remaining ethanol was allowed to evaporate. After multiplexing and purification, all DNA libraries were quantified using a NanoDrop spectrophotometer (NanoDrop Products, Wilmington, DE, USA) and then pooled in equal concentrations. Lastly, a Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) was used to verify product length. The pooled mixture was then diluted to 4pM with Tris and was placed onto the Illumina MiSeq V3 flow cell with 20% PhiX. The MiSeq instrument at the University of Tennessee Genomics Core, Knoxville, Tennessee, USA was set to read a 2x300 paired end cycle.