Gut Microbiome Profiling in Mice

Fecal microbiota samples were collected from duodenum, jejunum, ileum, cecum, and colon of 8-week old naive C56Bl/6 J mice after at least 1 week of acclimatization post mouse delivery. QIAamp Fast DNA Stool Mini Kit (Qiagen, cat#12855) was used DNA extracting. Amplicons spanning variable region 4 (V4) gene were generated with primers and barcodes (515 F, 806 R)^{52 (link)} using HotMaster Taq and HotMaster Mix (QuantaBio) and paired-end sequenced using Illumina MiSeq platform. The sequence was performed at the Harvard Medical School Biopolymer Facility. Established protocol for QIIME 2 software^{52 (link)} and LEfSe (Linear discriminant analysis Effect Size) version 1^{53 (link)} was used for data analysis. Sequences were quality filtered in which reads were truncated if two consecutive bases fall below a quality score of Q20 (1% error), and reads that were <75% of full length were discarded^{54 (link)}.