Interplay of the host local microbiota and genetic perturbations dictates site-specific development of intestinal neoplasms in mice
DNA Extraction, 16S rDNA Amplification, and Multiplex sequencing DNA was obtained from distal and proximal cecum using the DNeasy Blood and Tissue Kit (QIAGEN). Bacterial 16S rRNA genes were amplified using the primers as described in (Caporaso et al., 2012). Each sample was amplified in quadruplicate, combined, and cleaned using the Agencourt AMPure XP beads (Beckman Coulter, Inc.). PCR reactions contained 0.5 µM for each primer, 100 ng genomic DNA and Phusion High-Fidelity PCR Master Mix (NEB). Reactions were held at 98*C for 30s, proceeding to 35 cycles at 98*C for 10s, 50*C for 30s, and 72*C for 30s; a final extension of 10 min at 72*C. Cleaned amplicons were quantified using Picogreen dsDNA and a composite sample for sequencing was created by combining equimolar ratios of amplicons from the individual samples, agarose gel purified (QIAquick Gel Extraction, QIAGEN) and mixed with 50% PhiX (Illumina) and loaded on Illumina sequencer as previously described (Caporaso et al., 2012).