title: GSM6696843: Normal, replicate 2, scRNAseq; Homo sapiens; RNA-Seq
library_name: GSM6696843
library_construction_protocol: The fresh ascending aortic wall tissues were washed at least 3 times with the Hanks Balanced Salt Solution (HBSS) (14025092, Thermo Fisher Scientific, Waltham, MA, USA) to wash away the blood. The adipose tissue attached to the adventitia was removed by blunt separation using the forceps. The single cell preparation was performed on ice to preserve cell viability. The aortic tissues were cut into smaller pieces (1–2 mm3) and digested in a 15-ml centrifuge tube with a digestive enzyme cocktail that included 3 mg/ml Type I collagenase (LS004176, Worthington Biochemical Corp., Lakewood, NJ, USA), 0.156 mg/ml Type XI collagenase (H3506, Sigma Corp., Kanagawa, Japan), 0.25 mg/ml soybean trypsin inhibitor (LS003571, Worthington), 0.1875 mg/ml lyophilized elastase (LS002292, Worthington), 0.24 mg/ml Type I hyaluronidase (H3506, Sigma) and 60 U/ml DNase I (10104159001, Roche, Shanghai, China) as previously described. The samples were shaken slowly for 2-3h in a 37°C water bath to facilitate complete digestion. The digested supernatant was separated from the tissue remnants. The di-gestive reaction was stopped by adding Dulbecco's modified Eagle's medium (DMEM) and 10% fetal bovine serum. The remaining tissue was further digested with a fresh enzyme cocktail. The digestion process was repeated 3 times to ensure that all the cells from the aortic tissues were isolated. The cells were pelleted by centrifugation at 300 xg and 4℃ for 5min and resuspended in 1X PBS to generate a single cell suspension. The cells were counted using the TC20 automated cell counter (Singleron). The live cell counts were obtained by staining with trypan blue (302643, Sigma). The samples with >80% cell viability and 1×106 cells / ml were further processed for sequencing. Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.