GSM6576375
P14 mouse cerebella were homogenized in cold sucrose buffer (0.3 M sucrose, 5 mM HEPES, pH 7.4). The supernatant and the layer of myelin on the top of the pellet were collected by centrifugation of the cerebellar homogenate and discarded. The pellet was resuspended in sucrose buffer, and the large vasculature was separated from microvessels by centrifugation. Next, the supernatant was clarified by centrifugation. Finally, the micro-vasculatures were enriched in the pellets. The microvascular pellets were digested into single-cell suspension by 1 mg/mL collagenase/dispase (Roche, 10269638001) and 0.5 μL/mL benzonase (Merck Millipore, E1014) in Dulbecco's modified eagle medium (DMEM) (Corning, 10-017-CV) at 37 °C for 5–10 min. After erythrocytes were lysed on ice for 5 min, the single cell suspension was incubated with complex of anti-biotin microbeads (Miltenyi Biotec, 130-090-485) and conjugated with rat anti-PDGFRβ-biotin antibody (Thermo Fisher Scientific, 14-1402-82) at 4 °C for 45 min. Then, the supernatant containing pericyte-PDGFRβ antibody -magnetic microbead complexes were passed through MS column (Miltenyi Biotec, 130-042-201) to collect the pericytes. RNA-seq transcriptome library was prepared following TruSeqTM RNA sample preparation Kit from Illumina (San Diego, CA) using 1μg of total RNA. Shortly, messenger RNA was isolated according to polyA selection method by oligo(dT) beads and then fragmented by fragmentation buffer firstly. Secondly double-stranded cDNA was synthesized using a SuperScript double-stranded cDNA synthesis kit (Invitrogen, CA) with random hexamer primers (Illumina).