GSM4502447: Aortic_isograft_2wk 1; Mus musculus; RNA-Seq
Aortic isografts were harvested from the mice. Adventitial layer of the grafts was first dissected from the medial and neointimal layers. After three times washes of PBS, grafts were cut into fine pieces and digested in digestion solutions (which consist of 1 mg/ml papain, 0.5 mg/ml 1,4-Dithioerythritol, 0.156 mg/ml Taurine; 0.25 mg/ml bovine serum albumin in PBS) at 37 °C with reciprocal shaking for a total 40 min. Detached cells were then collected through a 40 μm cell strainer and suspended in DMEM with 10% FBS. Cells were further stained with LIVE/DEAD ™ Fixable Near-IR Dead Cell Stain Kit and Hoechst 33342 for 20 mins. After PBS washing, cells were resuspended in PBS and single nucleated live cells (Hoechst+/Dead Cell Stain-) were sorted into PBS with 0.04% BSA using a Flow Cytometer. Cells were then analyzed on a 10x Chromium instrument using Chromium Single Cell 3' Reagent Kit v3 chemistry (10X Genomics), following a standard protocol. Library was generated and sequenced on a NovaSeq platform (Illumina) with paired-end 150 bp (PE 150) sequencing strategy.