GSM8290878: Endothelial cells E10.5-12.5 C; Mus musculus; OTHER
Embryos were harvested at E18.5 and their lungs were isolated. For each induction, three independent cell preparations were processed. Three to four fetal lungs of male embryos were pooled in 750 µl of DMEM (Lonza) and mechanically dissociated them using dissection scissors. Following the addition of collagenase I, II, and IV (Sigma-Aldrich) at a final concentration of 5 µg/ml, the samples were incubated at 37°C in an Eppendorf shaker at 1000 rpm for 10 minutes. Subsequent mechanical dissociation involved vigorous resuspension, after which the cell suspensions were filtered through a 40-µm cell strainer. The filters were rinsed with 750 µl of DMEM with 10% FCS, and the resulting cell suspensions were centrifuged at 4°C for 10 minutes at 1000 rpm. After removing the supernatant, the cells were resuspended in 300 µl of 1x red blood cell lysis buffer at 4°C for 1 minute, diluted them in 900 µl of PBS with 10% FCS, and centrifuged them (10 minutes at 4°C, 1000 rpm). The cells were then resuspended in 300 µl of PBS with 5% FCS. Cells were counted and incubated at 4°C in the dark for 40 minutes with the following antibodies: CD31-PE-Cy7 (1:100, ThermoFisher), CD45-PE-Texas Red (1:100, ThermoFisher), CSPG4-Alexa-488 (1:100, Merck), and PDGFR-β-APC (1:100, ThermoFisher). Prior to sorting, the cells were concentrated and resuspended in 800 µl of PBS with 5% FCS. DAPI (1:10000, BD Biosciences). CD45- cells were sorted for CD31 and for CSPG4/PDGFRB using a BD FACS Aria III and BD FACSDiva software version 8.0.1. DNA was isolated from the sorted cells using QIAamp DNA micro kit according to the manufacturer's protocol, and RNA was isolated by Trizol extraction. At the E8.5-10.10 induction time point, cells were sorted using an unconjugated CSPG4 antibody (1:100, Millipore) for 40 minutes, and subsequently incubated with goat anti-rabbit Alexa 488 secondary antibody (1:200, Jackson) for 30 minutes. MeD-seq analyses were performed as previously described, with all experimental time points conducted in triplicate (25, 34).