title: GSM6456204: Aorta Control, scRNAseq; Mus musculus; RNA-Seq
library_name: GSM6456204
library_construction_protocol: Aortae from 4 control and 4 diabetic mice were collected in DMEM media containing 10 % fetal calf serum (FCS) on ice. Aortae were then individually cut into small pieces and digested for 1 hour at 37 °C on a thermomixer in PBS containing LiberaseTM (0.1 mg/mL, Sigma), 1.4 mM CaCl2, Hyaluronidase (60 U/mL Sigma), DNase I (Qiagen 120U/mL). The aortic suspension after digestion was filtered through a 40 µM cell strainer and washed with FCS containing DMEM media. Cells from the experimental groups (control and diabetes) were pooled and resuspended in the FCS containing DMEM media and incubated at 37 °C with 5% CO2 in a cell culture incubator for 30 minutes. Aortic cells were resuspended in FACS buffer containing 1X HBSS, 0.05 mM EDTA, and 5 g BSA and incubated with fluorochrome-coupled antibodies (7AAD-PE/Cy5 and Calcein-FITC) to sort viable and metabolically active cells using FACS Fusion (BD Biosciences). scRNA-seq libraries prepared according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, sorted aortic cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into  10X chip chip to generate droplets or gel bead in emulsions (GEMs). Poly-A RNA transcripts from lysed cells captured in GEMs were retrotranscribed to cDNA, and barcoded according to embedded bead functional oligos which contain an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. Barcoded cDNA was amplified by PCR, enzymatically fragmented and sized selected with SPRIselect reagent for subsequent library construction. During final library construction unique sample indexes were included from the 10X genomics "Single Index Kit T Set A".