Shotgun metagenomic sequencing of fasted and anesthetized mice

This experiment was conducted to elucidate the mechanisms underlying the characteristic changes in the gut microbiota during QIH, focusing on two major physiological changes associated with this state: fasting and hypothermia. To assess their individual contributions, wild-type mice were subjected to either food deprivation or anesthesia-induced hypothermia. The intestinal microbiota were analyzed at multiple time points (0, 18, 36, and 144 hours) after the onset of each condition. Genomic DNA was extracted from intestinal contents using the QIAamp PowerFecal Pro DNA Kit (Qiagen N.V., Hilden, Germany). Sequencing libraries were prepared with the NEBNext Ultra II FS DNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA) and NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 1 and Set 2). Sequencing was performed on a HiSeq system using a 2 Ã? 150 bp paired-end protocol (Azenta Life Sciences, Burlington, MA).