Analyzing Gut Microbiome in WT and Batf3-/- Mice

Fecal samples were collected from female and male 8-week-old WT and Batf3^-/- mice, snap-frozen, and stored at -80°C. DNA was isolated using the DNeasy PowerSoil DNA Isolation Kit (Qiagen) following the manufacturer’s protocol. The V4 region of the 16S gene was amplified and barcoded using 515f/806r primers and 250x2 bp sequencing was performed on an Illumina MiSeq system. Raw data were processed using DADA2 scripts in R platform and quality-filtered reads (~50,862 reads per sample) were used to identify amplicon sequence variants (ASV) by closed reference picking against the Silva database (54 (link)). Abundance of selected bacterial strains were confirmed by qPCR using 25 ng of fecal bacterial DNA and specific 16S rRNA primers for Mucispirillum schaedleri (ASF457), Akkermansia muciniphila, Bifidobacterium, Bacteroides sp. (ASF519) (primer sequences are listed in Supplemental Table). qPCRs were performed in duplicate using SsoAdvanced Universal SYBR® Green Supermix. Relative abundance for each strain was quantified by normalizing the quantity of each specific 16S rRNA gene to the total amount of 16S bacterial DNA. 16S rRNA sequences generated in this study are publicly available. This data can be found here: ENA, accession number PRJEB50182 (https://www.ebi.ac.uk/ena/browser/home).