Isolation and Preparation of Cisternal Milk Samples

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.