GSM5703459: CNC rep1; Mus musculus; RNA-Seq
Mice bearing a conditional CAG-Sun1/sfGFP (The Jackson Lab, 021039) tracer allele with either a Mef2cSHF (gifted by the K.R. Chien lab at the Cardiovascular Research Center, Massachusetts General Hospital, Boston, Massachusetts, USA) or Wnt1-Cre2 (The Jackson Laboratory, 003829) allele were sacrificed at 8 weeks of age. The aortic root and ascending aorta were dissected and cleaned with PBS. Minced tissue was transferred to ice-cold HBSS (Thermo Fisher Scientific, 14175095) containing 0.2 u/µL RNase inhibitor (Applied Biosystems, N8080119) and 5% D-trehalose (Quality Biosciences, A611-K480-95) and then digested with 1 mg/mL collagenase II(Worthington-Biochem, LS004176), 100ul elastase (MilliporeSigma, E1250), and 0.2mg/mL DNase I (Zymo Research, E1009) in a 37°C rotating water bath for 30 minutes with periodic agitation. After further trituration through a 21G needle, the digestion was stopped by addition of 10% Fetal Bovine Serum (FBS) (Corning Cellgro, 35-011-CV). Cells were pelleted by centrifugation, resuspended in the previously mentioned HBSS solution and passed through a 40 µm filter-cap tube. Cells were sorted using a DakoCytomation MoFlo at the Bloomberg Flow Cytometry and Immunology Core. Viability was determined by propidium iodide stain and GFP+ cells were sorted into a tube containing TriZol (ThermoFisher Scientific, 15596026). After separation by chloroform and addition of cold 70% ethanol, RNA was extracted using the PicoPure RNA Isolation Kit (ThermoFisher Scientific, KIT0204). Total bulk RNA was normalized according to sorted cell number, reverse transcribed and amplified according to the SmartSeq2 protocol used for single-cell sequencing (Picelli, et al. Nat. Protoc. 2014). The amplified cDNA was fragmented and barcoded using the Nextera XT DNA Library Preparation Kit (Illumina) and the resulting libraries were pooled.