16s rRNA gene of microbiota from small intestine of Swiss Webster Mus musculus conventionalized at day 21 or 22 for 21 days
Total DNA was isolated from 100 mg small intestinal content (SIC) of the conventionalized Swiss Webster. SIC was resuspended in 1 mL sterile PBS. Large debris was removed by centrifugation at 750 _ g for 5 min. Supernatants were then centrifuged at 10,000 _ g for 1 min to collect microbes. The pellets from SIC were resuspended in TE buffer containing Lysozyme (5 mg/mL) and incubated at 37 C for 45 min followed by 1 h at 56 C with 0.5 mg/mL proteinase K, before adding 1% (w/v) SDS and incubating for an additional 30 min at 37 C.  The DNA was extracted from the mixture and precipitated overnight with 3 M sodium acetate and washed with 70% ethanol. V4 region of 16S rRNA genes from extracted DNA were sequenced by Illumina Miseq.