GSM8125081: BAT and Artery; Mus musculus; RNA-Seq
The aortas (i.e., aortic arch to the iliac bifurcation) were isolated after perfusion with PBS and incubated with 10 mg/mL collagenase IV (Worthington) and 20 U/mL DNase I at 37°C for 40 min digestion, The interscapular brown adipose tissue (BAT) were collected, cut into small pieces and incubated with the digestion cocktail at 37°C for 30 min. The digested tissues were filtered through a 30-μm cell strainer and centrifuged to obtain single-cell suspensions. The single-cell suspensions were sorted using a BD FACSAria III Cell Sorter gated live CD45lowCD31highCD107ahigh cells as aortic endothelial cells, and live CD45-CD31+ as BAT endothelial cells. The sorted cells were deep-frozen in 2× TCL buffer (Qiagen) plus 1% beta-mercaptoethanol kept at -80°C until subsequent RNA extraction and library preparation. Libraries were then constructed with the NEB Next Ultra II FS kit (E6177S, NEB) using the prime-seq specifications