16s rRNA gene of microbiota cultured from small intestine and cecum (mixed in 1:1 ratio) from Swiss Webster Mus musculus conventionalized at day 21 or 22 for 21 days
Small Intestinal content (SIC) or cecal content (CC) from conventionalized Swiss Webster mice was weighed and resuspended in 1 mL sterile PBS. Large debris was removed by centrifuging the resuspension at 750 _ g for 5 min. The supernatant from this first low speed spin was collected and centrifuged with higher speed at 10,000 _ g for 1 min to pellet bacteria. The pellet was resuspended in 1 ml PBS per 1 g of original intestinal content or cecal content and inoculated by glass pipet into glass tubes containing 12 ml of semi-solid thioglycollate medium (Sigma). Tubes were sealed in ambient conditions before incubation at 37 C for 5 to 7 days. The bacteria cultured from SIC (SIB) or bacteria cultured from CC (CCB) were harvested by centrifugation at 10,000 _ g for 5 min. SIB or CCB were resuspended in 500 _L sterile PBS to reach O.D.600 =0.5. SIB and CCB were then mixed at 1:1 ratio from which 100 _L were collected for total DNA isolation. Supernatants were then centrifuged at 10,000 _ g for 1 min to collect microbes. The pellets 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.