title: GSM6604254: Mouse vessels, WT, main_rep1; Mus musculus; RNA-Seq
library_construction_protocol: Mice were euthanized by cervical dislocation and the head was immediately removed and placed in ice cold primary washing medium (Dulbecco's Modified Eagle Medium (DMEM) with 1% Penicillin/Streptomycin (P/S), both from Gibco. Once in the lab, the brain was carefully dissected from the skull and the brain stem and olfactory bulbs were removed. For scRNAseq, the cerebellum was also removed and only one hemisphere was used for further analysis. The brain was then placed in a 100 mm petri dish and minced by cutting with a pair of scissors parallel to the petri dish plane to avoid cutting into the plastic surface (<1 mm3 pieces). Once homogenous, the tissue was dissolved in 3 ml freshly prepared primary dissociation buffer (collagenase 1 mg/ml, Sigma C6885, in DMEM w/o phenol red, Gibco) and passed through a cut p1000 pipet tip ten times or until smooth passing. The solution was transferred to a 5 ml Eppendorf tube and placed in a rotating incubator (speed 20 rev/min) at 37 °C for 10 minutes. Samples were then passed through an uncut p1000 pipet tip 10 times or until smooth passing. Samples were placed back in the rotating incubator for 10 minutes at 37 °C. The samples were filtered through a 70 µm mesh strainer (Falcon) into a 50 µl tube (Falcon) to remove large undigested tissue fragments. The filter was washed with 10 ml of primary washing medium, and samples were spun at 300 g for 5 minutes at RT. The supernatant was removed, and the pellet was resuspended in 4.5 ml secondary washing medium (DMEM with 1% PS (Gibco), 0.5 mg/mL heparin (Sigma)) and divided into three 2 ml Eppendorf tubes. 15 µl Rat anti-mouse CD31 antibody (BD, 10 µl/brain) coupled Dynabeads® Sheep Anti-Rat IgG (Invitrogen, 50 µl/ brain, prepared according to manufacturer's instructions) were added to each subsample and placed in a rotating incubator for 30 minutes at room temperature. After antibody incubation, the tubes were placed on a DynaMag 2 magnet (Invitrogen), and the supernatant was removed. Beads were washed twice by resuspension and magnetic capture in secondary washing medium, after which the three samples were pooled in one 2 ml Eppendorf tube. The resuspended beads coupled with microvasculature were visualized using an inverted microscope at 20x magnification. Libraries were constructed as per manufacurers' recommendation (10x Genomics Gene expression v3.1)