title: 16S rRNA amplicon sequencing
design: DNA was extracted from approximately 250 mg cecal content, and 100 mg colonic content using the DNeasy PowerLyzer PowerSoil kit (Qiagen, 12855-100), using two-four blank DNA extraction controls. The V3 region of the 16S rRNA gene was PCR-amplified using 0.2 l Phusion High-Fidelity DNA polymerase (ThermoFisher Scientific, F-553L), 4 l HF-buffer, 0.4 l dNTP (10 mM of each base), 1 M forward primer (PBU; 5-A-adapter-TCAG-barcode-CCTACGGGAGGCAGCAG-3) and 1 M reverse primer (PBR; 5-trP1-adapter-ATTACCGCGGCTGCTGG-3) and extracted DNA diluted to 5 ng/l in a 20 l total reaction volume, with a PCR program consisting of initial denaturation for 30s at 98 C, followed by 30 cycles of 98 C for 15 s and 72C for 30 s, and lastly 72 C for 5 min to allow final extension before cooling to 4 C.  A total of two no-template controls as well as at least the two DNA extraction controls were included per PCR run. The PCR products were purified using the HighPrepTM PCR Magnetic Beads (MAGBIO, AC-60005) with a 96-well magnet stand (MAGBIO, MyMag 96), according to the manufacturers recommendations. DNA quantity was measured using Qubit dsDNA HS assay (InvitrogenTM, Q32851) and samples were pooled to obtain equimolar libraries and sequenced on the Ion S5 System (ThermoFisher Scientific) using Ion OneTouch 2 with the 520 chip kit-OT2 (ThermoFisher Scientific, A27751).