title: Milk exosome-derived miRNAs from cow
design: Approximate 40 mL raw milk samples were subjected to 2 successive centrifugations at 2,000 and 12,000  g at 4C for 30 min to remove somatic cells and debris. The supernatant was mixed with an equal volume of 0.25 M EDTA (pH 7.0) and incubated on ice for 15 min to precipitate casein and exosomes coated with casein. The suspension was ultracentrifuged at 80,000 g at 4C for 60 min (Sorvall WX Ultra 80, F37L-8  100 rotor; Thermo Scientific) to remove precipitated protein, milk fat globules, and microvesicles larger than exosomes. Exosomes were collected by centrifugation at 120,000 g at 4C for 90 min, resuspended in sterile phosphate-buffered saline, and filtered through a 0.22-m membrane filter to obtain exosome solutions. Then, exosomal RNAs from each milk sample were used for library construction and subjected to single-end sequencing in the length of 40 nt with an Illumina Genome Analyzer.