title: 16S of mus musculus: gut microbiome
design: Samples were kept at room temperature and upon arrival to the laboratory, further homogenized by vortexing and then stored at 4C. For DNA extraction, 400ul of the fecal suspension was transferred to the bead tubes supplied in the QIAamp PowerFecal Pro DNA Kit. Bead beating was performed for 1 min at 6.5 m/s on a FastPrep-24 system. DNA isolation then continued following the kit manufacturers instructions. 16S rRNA amplicon sequencing was performed on a MinION nanopore sequencer. The amplicon library was prepared using the 16S Barcoding Kit 1-24 (SQK-16S024). For the PCR amplification and barcoding, 15 ng of template DNA extracted from fecal samples. Initial denaturation at 95C was followed by 35 cycles of 20 s at 95C, 30 s at 55C, 2 min at 65C, and a final extension step of 5 min at 65C.  Purification of the barcoded amplicons was performed using the AMPure XP Beads as per Nanopores instructions. Samples were then quantified using Qubit fluorometer and pooled in equimolar ratio to a total of 50-100 ng in 10 l. The pooled library was then loaded into an R9.4.1 flow cell and run as per the manufacturers instructions. The MINKNOW software 19.12.5 was used for data acquisition.