GSM5068998: Single nucleus human perivascuar adipose tissue 3; Homo sapiens; RNA-Seq
For single cell, Aorta were isolated from mice, minced and placed into digestion medium (DMEM, Collagenase D: 6.1mg/ml (Roche), Dispase II: 2.4 mg/ml (Roche) and placed at 37oC with constant agitation at 200 rpm. To enrich for cell populations with differential sensitivity to digestion, we utilized the following procedures. For embryonic/newborn mice, 20% of the digestion was quenched at 15 minutes, 20% at 20 minutes and the remaining 60% at 25 minutes (embryos/perinatal). For adult aortas 50% of the digestion was quenched at 30 minutes, 50% at 60 minutes. Single cells were isolated from dissected aortas and flow sorted to isolate live (DAPI-) cells and remove debris. Cells were collected as either CD45+ (immune) and CD45- (non-immune) cells. For adult single cell RNA Sequencing, CD45+ cells were mixed with CD45- cells to achieve a proportion of ~20%. For single nuclei experiments, 40-80 mg of frozen human PVAT was dounce homogenized using a glass homogenizer (Sigma-Aldrich, 7 mL) on ice in 2.5 mL homogenization buffer (0.32 M sucrose (Sigma, RNase & DNase free ultra pure grade), 5 mM CaCl2 (Sigma), 3 mM MgAc2 (Sigma-Aldrich), 10 mM Tris-HCl pH 8.0 (FisherChem), 0.1% TritonX-100 (Amresco), 0.1 mM EDTA (Fischer), protease inhibitor cocktail (Roche)) and layered into ultracentrifuge tubes (Beckman Coulter) over 12 mL sucrose cushion (1.8 M sucrose, 10 mM Tris-HCl pH 8.0 (FisherChem), 3 mM MgAc2 (Sigma-Aldrich), protease inhibitor cocktail (Roche) and topped with 14.5mL homogenization buffer. Samples were ultracentrifuged (Beckman Coulter L-70K) using a swinging bucket rotor (Beckman Coulter SW28) at 25,000 rpm for 2 hrs at 4°C. The supernatant was aspirated to remove debris and the nuclear pellet was resuspended in 1 mL of DPBS (Corning) with RNase inhibitor (Lucigen). Samples were filtered and nuclei were counted before loading for snRNA-Seq For single cell experiments, single cell transcriptomes were collected  previously using the 10X Genomics platform with v2 (perinatal) and v3 (adult) chemistry (10× Genomics, Pleasanton, CA) and sequenced on Illumina HiSeq 4000. For single nucleus experiments, single nucleus transcriptomes were collected  using the 10X Genomics platform  v3 (adult) chemistry (10× Genomics, Pleasanton, CA) and sequenced on Illumina NovaSeq 6000.