RNA-Seq of mus musculus
Brain were collected for isolation of cerebral vessels. Briefly, mice were transcardially perfused with ice-cold phosphate-buffered saline. Then, brains were removed, rinsed in cold isotonic sucrose buffer (0.32 mol/L sucrose, 3 mmol/L HEPES, pH 7.4), cleared of pia mater and choroid plexus. The brain was homogenized with a tight fitting pestle Dounce homogenizer in 5 ml of sucrose buffer. The homogenate was centrifuged at 1000g for 10 min. The supernatant containing neuronal cells was discarded and the white layer of myelin in the upper part of the pellet was removed. The pellet was resuspended again in 5 ml of the same cold buffer and centrifuged at 1000g for another 10 min. We repeated the washes three times to eliminate the rest of the myelin and free neurons. The pellet was resuspended in 3 ml of the sucrose buffer and centrifuged at 350g to eliminate detached cells. This step was repeated three times and the resulting pellet was resuspended in 1 ml of sucrose buffer and centrifuged at 75g for 5 min. The resulting supernatant was used as the cerebral vessels preparation. RNA libraries were prepared for sequencing using standard Illumina protocols.