Fecal collection and isopropanol DNA extraction

Six to eight week-old mice were placed in an empty autoclaved cage and two freshly evacuated fecal pellets were placed in a sterile 2 ml Eppendorf round-bottom tube (Thermo Fisher Scientific, Pittsburgh, PA) containing a sterile 0.5 cm diameter stainless steel bead as previously described²⁰. Samples were stored at −80 °C until processed for DNA extraction. For DNA extraction, lysis buffer was added (800 µl per tube) and samples were mechanically disrupted using a TissueLyser II (Qiagen, Venlo, Netherlands) for 3 minutes at 30 Hz, followed by incubation at 70 °C for 20 minutes with periodic vortexing. Samples were centrifuged at 5000 × g for 5 minutes, and the supernatant was transferred to a sterile 1.5 ml Eppendorf tube containing 200 µl of 10 mM ammonium acetate. Lysates were vortexed, incubated on ice for 5 minutes, and then centrifuged. Supernatant was transferred to a sterile 1.5 ml Eppendorf tube and one volume of chilled isopropanol was added. Samples were incubated on ice for 30 minutes and centrifuged at 16000 × g at 4 °C for 15 minutes. The DNA pellet was washed with 70% ethanol and resuspended in 150 µl Tris-EDTA, followed by addition of 15 µl of proteinase K and 200 µl AL buffer (DNeasy Blood and Tissue kit, Qiagen). Samples were incubated at 70 °C for 10 minutes and 200 µl of 100% ethanol was added to the tubes. Samples were mixed by gentle pipetting and the contents transferred to a spin column from the DNeasy kit (Qiagen). The DNA was further purified following the manufacturer’s instructions and eluted in 200 µl EB buffer (Qiagen). DNA concentrations were determined fluorometrically (Qubit dsDNA BR assay, Life Technologies, Carlsbad CA) and samples were stored at 20 °C until sequencing.