GSM2413210: 11178X13; mouse gut metagenome; OTHER
Great care was taken to avoid microbial variability that is independent of experimental design.  Since the microbiome is transmitted from the dam, female C57BL/6J mice were newly purchased from Jackson labs as dams of all animals in the experiment, Further, animals from each genotype were housed together until weaning, then placed within their respective environment, to avoid non-genetic or environmental effects (Moon et al., 2015). Given the possible confounding effects of coprophagia, each cage contained similar numbers of animals from all 4 genotypes: wild-type, Tcf4Het/+ Apc+/+, Tcf4+/+ ApcMin/+, and Tcf4Het/+ ApcMin/+. Stool was collected from all mice at time of sacrifice from colon during dissection. Genomic DNA was isolated from control and enriched mouse stool samples using QIAamp DNA Stool Minikit (QIAGEN, 51504) following manufacturer’s instructions for stool pathogen detection protocol. Primers were designed to selectively amplify the V1-V3 bacterial genomic 16S rRNA regions and include adapter sequences compatible with the Nextera XT 96 Index Kit by Illumina. The two primer sequences chosen have been published previously as Bosshard forward and 533 reverse (Table S5; (Bosshard et al., 2002; Chen et al., 2012). Samples were amplified using 2x KAPA HiFi HotStart Ready Mix (KAPA Biosystems, KK2601) with primers at a final working concentration of 1 µM, purified using Agencourt AMPure XP beads (Beckman Coulter Genomics, A63880), and indexed using Nextera XT 96 Index Kit (Illumina, FC-131-1002). Final samples were re-purified using Agencourt AMPure XP beads (Beckman Coulter Genomics, A63880), resuspended in 10 mM Tris, pH 8.5, and pooled at a final concentration of 4 nM.