PCR-16s of O. franksi tissue
DNA extractions including negative controls were performed using the Qiagen Powersoil DNA Isolation Kit following the standard protocol. Extractions were eluted using 200ul AE buffer and concentrations were quantified using gel electrophoresis (5ul sample 1.5% agarose) and a Nanodrop 2000 Spectrophotometer (Thermo Fisher Scientific). Extracted DNA varied in their final concentrations from 1.2-50ng/ul out of 20ul aliquots. Two-stage amplicon PCR was performed on the V4 region of the 16S rRNA marker gene to target bacterial and archaeal communities. We used the 515F (5-ACA CTG ACG ACA TGG TTC TAC AGT GCC AGC MGC CGC GGT AA-3) and 806R primers (5- TAC GGT AGC AGA GAC TTG GTC TGG ACT ACH VGG GTW TCT AAT -3), with the addition of common sequence 1 and 2 linkers at the 5 end. These linkers are required for Illumina MiSeq amplicon tagging and indexing. PCR were prepared with 9 L 5Prime HotMaster Mix (VWR International), 8 L of PCR-grade water, 1 L forward primer (10 M), 1 L reverse primer (10 M), and 1 L of template DNA We used the following conditions for the PCR: 3 min of denaturation at 94 C; 30 cycles of 45 s at 94 C, 60 s at 50 C, and 90 s at 72 C; then finally 10 min at 72 C. Samples were then held at 4 C before sequencing. Sequencing preparation for amplicon libraries was performed at Rush Core Sequencing facility at the University of Chicago (Chicago, IL, United States) using two lanes of an Illumina Miseq chip.