Mouse gut microbiome following temperature conditioning

Adult male C57BL/6J mice were obtained from The Jackson Laboratory and housed at 22 degrees C for 1 w prior to temperature acclimation. To prepare 30 degree Celsius-conditioned gut microbiota for transplantation, mice (n = 8) were housed at 30 degrees Celsius for 3 w. For 22 degree Celsius- and 4 degree Celsius-conditioned gut microbiota, mice (n = 4-5) were similarly housed at 30 degrees Celsius for 3 w then transferred to either 22 degrees Celsius or 4 degrees Celsius for 4 w. All mice were housed 1 mouse/cage and euthanized at 12 w of age. The cecum and colon were placed in anaerobic PBS on ice immediately following excision. Within a Coy Chamber (anaerobic environment), 1-2 fecal pellets from colons of individual mice were placed in a sterile microfuge tubes and kept on ice, and the remaining contents of the cecum and colon from all mice were combined in anaerobic PBS (3mL per mouse) with 10% glycerol. The cecal/fecal slurry was vortex mixed, centrifuged at 400xg for 3 min, and the pellet was discarded. The remaining donor homogenate was aliquoted and placed at -80 degrees Celsius. To perform fecal microbiome transplantation (FMT), recipient mice were administered an antibiotic cocktail ad libitum for 48 h. 6 h following antibiotic withdrawal, recipient mice received 100 microliters of respective donor homogenate via oral gavage, which was repeated daily for 4 sequential d. Recipient fecal pellets (2 w following initiation of FMT) were collected fresh on ice and stored at -80 degrees Celsius, as were the donor fecal pellets and homogenates (collected at time of preparation), until later sequencing.