16s rRNA gene of microbiota from small intestine of SPF 129S1/SvImJ Mus musculus cohousing with the germ free Swiss Webster Mus musculus
Total DNA was isolated from 100 mg small intestinal content (SIC) of the SPF 129S1/SvImJ Mus musculus cohoused with the germ free Swiss Webster Mus musculus. 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.