Comparison of T5KO and wildtype mice
To avoid the confounding effects of cohousing on the diversity of cecal bacteria, we selected mice from multiple litters that were housed separately. Bulk DNA was extracted from frozen extruded cecal contents using a modified phenol-chloroform extraction technique with mechanical disruption (bead-beating). Briefly, an aliquot (~100mg) of each homogenized sample was, while frozen, suspended in a solution containing 500 ul of DNA extraction buffer [200 mM Tris (pH 8.0), 200 mM NaCl, 20 mM EDTA], 210 ul of 20% SDS, 500 ul of a mixture of phenol:chloroform:isoamyl alcohol (25:24:1)], and 500 ul of a slurry of 0.1-mm-diameter zirconia/silica beads (BioSpec Products, Bartlesville, OK). Microbial cells were then lysed by mechanical disruption with a bead beater (BioSpec Products) set on high for 2 min (23C), followed by extraction with phenol:chloroform:isoamyl alcohol, and precipitation with isopropanol. DNA was quantified using the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen) and a plate reader. For each sample, 16S rRNA genes were amplified using a composite forward primer and a reverse primer containing a unique 12-base barcode used to tag each PCR product, as previously described. Thus, for each sample, we amplified the 16S rRNA gene using the composite forward primer 5' GCCTTGCCAGCCCGCTCAGTCAGAGTTTGATCCTGGCTCAG- 3': the italicized sequence is 454 Life Sciences primer B, and the bold sequence is the broadly conserved bacterial primer 27F. The reverse primer used was 5' GCCTCCCTCGCGCCATCAGNNNNNNNNNNNNCATGCTGCCTCCCGTAGGAGT-3': the italicized sequence is 454 Life SciencesÃ¢ primer A, and the bold sequence is the broad- range bacterial primer 338R. NNNNNNNN designates the unique twelve-base barcode used to tag each PCR product, with 'CA' inserted as a linker between the barcode and rRNA primer. Three independent PCRs were performed for each sample along with a no-template control. For each sample, the three replicate PCR products were combined and purified with Ampure magnetic purification beads (Agencourt Bioscience Corp. Beverly, MA), and products quantified described above. A master DNA pool was generated from the purified products in equimolar ratios to a final concentration of 21.5 ng/ul. Negative controls (both no-template and template from unused swabs) were included in all steps of the process in order to check for primer or sample DNA contamination. All aliquoting and diluting of  primers, as well as assembly of PCR reactions, was done in a PCR hood in which all surfaces and pipettors had been decontaminated with DNA Away (Molecular BioProducts, San Diego, CA, USA) and exposed to ultraviolet light for 30 min.  