Hotmaster taq

PCR was used to amplify the ITS2 region using the fITS7 [38] and ITS4 [39] primers. The forward primer sequence (fITS7) used was GTGARTCATCGAATCTTTG, and the reverse primer sequence (ITS4), was TCCTCCGCTTATTGATATGC. The PCR conditions included 0.5 U HotMaster Taq (QuantaBio) in a 25 μl volume using 10 x HotMaster Taq Buffer, 200 μM of each dNTP (Promega), 0.2 μM of primer and 4 μL of DNA extract. The thermocycling conditions consisted of an initial enzyme activation step at 94°C for 2 min, followed by 35 cycles of denaturation at 94°C for 20 s, annealing at 55°C for 10 s and elongation at 65°C for 40 s, with a final extension at 65°C for 10 min. Each set of PCRs included extraction and PCR blanks. All PCR products were visually examined by electrophoresis on 2.0% agarose TAE gels.

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)}.