3046-16S_For_bc1004-16S_Rev_bc1045.fastq
Fecal samples were collected as previously described [56, 59]. Individual mice were placed in sterile unbedded cages and allowed to defecate, fecal samples collected, and stored at -80C. DNA was extracted using the QIAamp PowerFecal Pro DNA extraction kit (Qiagen, USA) and assessed via Nanodrop, Qubit, and agarose gel electrophoresis. Library construction and sequencing were performed at the Roy J. Carver Biotechnology Center, University of Illinois at Urbana-Champaign. Full length 16S (FL16S) amplicons were created using 2.5ng of genomic DNA according to the published PacBio 16S protocol. Briefly, PCR reactions were set up on ice in 25 l volumes using barcoded primers with the following reagents: 12.5 l Kappa Hotstart Ready Mix  (KAPA Biosystems), 1.5 l water, 3 l (2.5 M) 16S Forward primer (AGRGTTYGATYMTGGCTCAG), 3 l (2.5 M) 16S Reverse primer (RGYTACCTTGTTACGACTT), 5 l DNA (0.5 ng/l). PCR cycling parameters were: denature 95C for 3 minutes, 25 cycles of 95C for 30 seconds, 57C for 30 seconds, 72C for 60 seconds, and hold at 4C. All products were measured with SpectraMaxQuant AccuClear Nano kit and run on an Agilent Fragment Analyzer for QC.  Samples were pooled according to concentration and cleaned twice with 0.6 volumes of magnetic beads. The cleaned pool was checked on an Agilent Fragment Analyzer before library production. Amplicons were converted to a library with the SMRTBell Express Template Prep kit 2.0 (Pacific Biosciences, Menlo Park, CA). The library was sequenced on 1 SMRTCell 8M on a PacBio Sequel IIe using the CCS sequencing mode and a 15hs movie time. CCS analysis was done using SMRTLink V10.0 using the following parameters: ccs --min-passes 3 --min-rq 0.999, lima --ccs --different --split-bam-named. Demultiplexing of the PacBio library was done with SMRT link V11 (Pacific Biosciences, Menlo Park, CA).