Sample collection, processing and shipment
BLOOD, STERILE
Blood, urine, and fecal samples were collected in our metabolic unit using standard protocols (Translational Research Institute, Orlando, FL, USA). Fecal samples were collected each time they were produced during the 6-day measurement periods in the whole room calorimeter. Sample weights were tracked upon collection and as aliquots were prepared. Fecal samples were processed within an hour of production under an anaerobic hood and were maintained on ice during processing. After mixing with a sterile spatula, samples were sub-aliquoted for various downstream applications. Samples for metagenomic sequencing and SCFAs were snap frozen without additives and stored at −80 °C. They were shipped overnight on dry ice. Any fecal sample not needed for method-specific aliquots were stored (sealed) in the original collection container at −20 °C within 60 min of collection. At the end of each 6-day calorimetry period, all collection containers were opened, and all frozen samples were transferred into a single, large, homogenization container to create a composite sample (without additives) that was used to measure fecal energy and biomass. The composite was partially thawed on ice while remaining sealed and then homogenized, on ice, using a sterile paddle homogenizer. The composite sample was stored at −80 °C until used or shipped overnight on dry ice