GSM9465244: Microvasculature_43WT_rep1; Mus musculus; RNA-Seq
Brain halves were mechanically and enzymatically digested with Collagenase type II (1 mg/mL, cat. #C6885, Sigma). On ice, the washed microvascular fragments were isolated using sheep anti-rat magnetic Dynabeads (50 mL/brain, cat. #11035, Invitrogen) bound to rat anti-mouse PECAM1 antibody (10 mL/brain, cat. #553370, BD Biosciences) and a DynaMag 2 magnet (Invitrogen). The washed microvessels were then further dissociated with Collagenase IV (1 mg/mL, cat. #17104-019, Gibco) and TrypLE (cat. #A1217701, Gibco) to remove the magnetic beads and obtain single cell solutions. Isolated microvascular fragments (n=2) were dissociated into single-cell suspensions, the viability and cell count was determined using LUNA-FLÔ Dual Fluorescence cell counter (Logos Biosystems). All samples showed above 92% cell viability. The single-cell suspensions were processed using Next Gem Single Cell 3' Reagent kit v3.1 with dual indexes (10X Genomics). The samples were prepared to capture ~5000 cells per sample and loaded according to the manufacturer's recommendations. The prepared cDNA libraries were subsequently sequences on a Next seq200 sequencer equipped with a P3 100 cycles flow cell.