GSM6896558: Ang_A; Mus musculus; RNA-Seq
Mice were euthanized and perfused with PBS and aortas were harvested and placed in a petri dish containing DMEM with 10% fetal bovine serum (FBS) on ice. Perivascular adipose tissues and connective tissues were carefully removed. After collecting all grafts, to obtain single cell suspensions, arteries were washed with PBS for three times, cut into fine pieces and incubated with 1 mg/ml papain digesting solution [1 mg/ml papain (Sigma, P4762), 0.5 mg/ml 1,4-Dithioerythritol (Sigma, D8255), 0.156 mg/ml Taurine (Sigma, T8691), 0.25 mg/ml bovine serum albumin in PBS]. The digestion was performed at 37 ℃ water bath with reciprocal shaking. Detached cells in the supernatant were collected and suspended in DMEM with 10% FBS. Vascular allografts were then incubated with fresh digestion solutions every 10 min. After 3-4 digestions, the remaining tissues were incubated with 1 mg/ml papain digesting solution and 2 mg/ml collagenase I (Gibco, 17018029) and 1 mg/ml dispase II (Sigma, D4693) in Hank's balanced salt solution containing calcium and magnesium. Detached cells were collected and fresh digestion solutions were added until the tissues dissolved completely. All digested cells from one sample were pooled together at the end of the digestion process. After sufficient digestion, cells were filtered through a 40-μm cell strainer, and the collected cells were centrifuged 8 min at 500 g speed at 4 ℃. Subsequently, cell pellet was suspended in PBS and stained with LIVE/DEAD ™ Fixable Near-IR Dead Cell Stain Kit, Hoechst 33342  for 30 mins on ice. 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. Samples were subjected to scRNA-seq. A ChromiumTM Single Cell 3' Reagent Kit v2 or V3 chemistry (10x Genomics) was used and a standard protocol was followed. The library was generated and sequenced on a Novaseq6000 PE150 platform (Illumina) with paired-end 150 bp sequencing strategy. RNA libraries were prepared for sequencing using standard Illumina protocols