DNA Extraction
Total DNA, including microbial genomic DNA, was extracted from fecal
pellets using the QIAmp PowerFecal Pro DNA Kit (Qiagen, Germantown, MD)
according to the manufacturer’s protocol with minor modifications as
follows. Each pellet was resuspended in 800 ul of Solution CD1, after
which samples were homogenized with 3 runs of 30 s at 6 m/s in a
FastPrep-24 5G bead beater (MP Biomedicals, Solon, OH). Following
centrifugation at 15,000 x g for 1 min, the supernatant was transferred
to a clean tube and 200 ul of Solution CD2 was added. After another
centrifugation at 15,000 x g for 1 min, the supernatant was transferred
to a clean tube and mixed with 600 ul of Solution CD3. The resulting
lysate was spun through an MB Spin Column and the flow-through
discarded. Columns were washed with 500 ul of Solution EA and then 500
ul of Solution C5. Following a drying step at 16,000 x g for 2 min,
50-100 ul of Solution C6 was added to elute the DNA. Buffer-only
negative controls were processed identically in parallel. DNA
concentrations and quality were determined using a NanoDrop 1000 (Thermo
Scientific, Waltham, MA), and DNA was stored at -20C until 16S library
preparation.