GSM8207659: brain vessels, western diet, rep3; Sus scrofa; RNA-Seq
Blood microvessels were isolated from the right brain temporal region using an established method.This method is designed to purify neurovascular units which comprise small blood vessel and associated pericytes and perivascular astrocyte end-feet. Briefly, a 150-200 mg frozen slice of the temporal region was minced in sterile HBSS, 10 mM HEPES (buffer B1),  homogenized in a Dounce homogenizer, and centrifuged at 2,000 g for 10 min at 4 °C. The pellet, comprising microvessels and myelin, was resuspended in ice-cold buffer B2 (B1 containing 18 g dextran from Leuconostoc spp/100 mL). After centrifugation at 4,400 g for 15 min at 4 °C, the supernatant and myelin were discarded and the vessel pellet resuspended in ice-cold buffer B3 (B1 containing 1% BSA), filtered through a 20 µm mesh membrane (Millipore, Burlington, MA), and recentrifuged. The pellet, containing purified brain microvessels was immediately processed for RNA isolation. Libraries were constructed using Illumina TruSeq RNA Sample Preparation Kit v2 (Illumina, San Diego, CA, USA) and AMPure XP beads (Beckman Coulter, Hercules, CA) according to the manufacturers' protocols. Library quantification was performed using KAPA Library Quantification kit (KAPA Biosystems, Wilmington, MA) and library fragment size was determined using Experion DNA 1K Analysis kit (Bio-Rad, Hercules, CA).