16S Sequencing of antibiotic-treated SPF, healthy-colonized, and CMA-colonized mice

DNA was extracted using the QIAamp PowerFecal Pro DNA kit (Qiagen). Samples were suspended in PowerBead tubes along with lysis buffer and loaded on a Bead Mill 24 Homogenizer (Thermo Fisher) or taped to a vortex. Tubes were agitated at maximum speed for 10 minutes at RT to dissociate the fecal pellet. PowerBead tubes were then centrifuged at 10,000 x g for 30 seconds at RT. Isolation of released DNA was then conducted according to the protocol of the manufacturer. DNA was purified routinely using a spin column filter membrane and quantified using the Qubit 4 Fluorometer (Thermo Fisher). Ileal contents were subjected to DNA isolation using the PowerSoil DNA Isolation Kit (MoBio) or the QIAamp PowerFecal Pro DNA kit (Qiagen), according to the protocol of the manufacturer.Isolated DNA was sequenced at the Argonne National Laboratory Sequencing Facility or the Duchossois Family Institute (DFI) at the University of Chicago. For samples sequenced at Argonne, 16S rRNA gene amplicon sequencing was performed on an Illumina MiSeq instrument. Paired-end reads (151 bp) were generated with 12-bp barcodes. The V4 region of the 16S rRNA gene was amplified using 515F and 806R primers that included sequencer adapter sequences used in the flow cell.Experimental details are described in Campbell et al.