title: Sample 235
design: Fecal DNA was extracted using the DNeasy PowerSoil Kit (SPF samples) or DNeasy PowerSoil HTP 96 Kit (384) (ASF samples). For mucosal samples, approximately 1 cm pieces of proximal colon were stored in TRIzol Reagent at -80C. After samples were thawed, they were rinsed in PBS and added to the bead tube of the DNeasy PowerSoil Kit (Qiagen). 60 L of Solution C1 (Qiagen DNeasy PowerSoil Kit) were added to each tube, and the tubes were incubated at 70C for 10 minutes. Samples underwent bead beating in a Qiagen TissueLyser II at setting 30 for 2 minutes. 20 L of proteinase K (Sigma) were added to each tube, and tubes were incubated at 65C for 30 minutes. Samples were then processed as per the DNeasy PowerSoil Kit (Qiagen) protocol beginning after the vortexing step.  The 16S rRNA V4 region was PCR-amplified using primers adapted from the 515F and 806R primers used by the Earth Microbiome Project and modified to include Illumina paired-end adaptors (forward: CTT TCC CTA CAC GAC GCT CTT CCG ATC TGT GCC AGC MGC CGC GGT AA; reverse GGA GTT CAG ACG TGT GCT CTT CCG ATC TGG ACT ACH VGG GTW TCT AAT) [65]. Nextera XT indices (Illumina) were attached to the 16S V4 amplicons during a second PCR step. Both PCR steps were performed using 5PRIME HotMasterMix (Quantabio). Cycling conditions for the first step were: 94C for 3 minutes; 20 cycles of 94C for 45 seconds (28 cycles for mucosal samples), 50C for 60 seconds, and 72C for 90 seconds; then 72C for 10 minutes. For mucosal samples, two separate 16S rRNA PCR reactions were performed for each sample, and the products from the two independent first round PCR reactions were combined as input for the second PCR round to attach indices. Cycling conditions for the second step were: 94C for 3 minutes; 5 cycles of 94C for 45 seconds, 65C for 60 seconds, and 72C for 90 seconds; then 72C for 10 minutes. Amplicons were purified and normalized using the SequalPrep Normalization Plate Kit (Invitrogen). Paired-end sequencing was done on the Miseq platform using the 300-cycle V2 kit for stool samples and the 300-cycle Micro kit for mucosal samples.