2.0 ml tube

Cisternal MS with a content of 45 mL stored at −80 °C were thawed overnight at 4 °C. Samples were then prepared according to the protocol provided by Siebert et al. [39 (link)]. First, 3.0 mL 0.5 M EDTA and 2.0 mL TBE buffer were added to the CMS, which were mixed carefully. Thereafter, samples were centrifuged at 13,000× g at 4 °C for 20 min to divide the skim milk and milk fat fraction. The milk fat fraction was removed, and the skim milk was reduced to 1 mL by removing the supernatant and carefully mixed to resuspend the pellet. This suspension was then transferred into a 2.0 mL tube (Eppendorf SE, Hamburg, Germany) and centrifuged at 16,000× g for one minute at room temperature. The supernatant was discarded until only 400 µL remained. A total of 100 µL was removed and transferred to a new 2.0 mL tube (Eppendorf SE). All udder quarters of one cow were then pooled to a volume of 400 µL. This FUQS was then transferred into a 2.0 mL lysing matrix tube E (MP Biomedicals) and homogenized using the FastPrep-24™ device (MP Biomedicals) as described for TCS. All further steps were conducted according to the TCS described above.

After sacrifice, the mice were weighed and positioned under a laminar flow for dissection. Samples were collected from the digesta of the fundus, the ileum approximately 1 cm before the cecum, from the cecum at the apex, and the colon approximately 1 cm after the cecum. The digesta was transferred into a 2.0 mL tube (Eppendorf AG, Hamburg, Germany) containing 600 µL of stool DNA stabilizer (Invitek Molecular GmbH, Berlin, Germany). Tubes were then stored immediately at −80 °C.

Twenty seeds were used to obtain a seed extract sample in the following procedure: The seeds were pulverized with beads in a 2.0 mL-tube (Eppendorf) using an automatic grinder at 2800 rpm for 3 min. Seed extraction was performed by shaking the pulverized sample with 50 µL of ultrapure water per mg of the sample at room temperature for 120 min under dark conditions. The seed extract was filtered using a membrane filter to remove the particles.