GSM7033658: Control mouse endothelial cells rep 2; Mus musculus; RNA-Seq
MoEC overexpressing or knockdown for NOVA2 were cultured in DMEM-high glucose with 1mM sodium pyruvate (Euroclone Cat# ECB7501L) supplemented with 10% FBS (Euroclone Cat# ECS500L), 2 mM L-glutamine (Euroclone, Cat# ECB3000D), 100 U/L penicillin/streptomycin (Euroclone Cat# #ECB3001D), 25 mM HEPES (Euroclone, Cat# #ECM0180), 100 g/mL heparin (from porcine intestinal mucosa, Merck Millipore Cat# H3149), 5 μg/mL EC growth supplement (ECGS from bovine pituitary gland, Merck Millipore Cat# E2759) under puromycin selection (3 μg/ml, InvivoGen Cat# ant-pr-1). Since NOVA2 expression is regulated by EC density, for the splicing analysis of NOVA2 targets moEC overexpressing HA-tagged NOVA2 were grown as sparse (500.000 cells in 100 mm Petri dishes), while NOVA2-knockdown moEC were seeded as confluent monolayers (500.000 cells in 35 mm Petri dishes). Total RNA was isolated using the RNeasy Mini Kit (QIAGEN) according to the manufacturer's instructions. After treatment with DNase (Ambion), 2–3 μg of RNA was retro-transcribed with mix of d(T)18 oligos and random hexamers or gene-specific primers (Supplementary Table 10) and Superscript III RT (Invitrogen)