title: NextSeq 500 sequencing; Single cell transcriptional profiling of aortic endothelium identifies a hierarchy from endovascular progenitors to differentiated cells
design: Single cell transcriptional profiling of aortic endothelium identifies a hierarchy from endovascular progenitors to differentiated cells
library_construction_protocol: Aortas were dissected and digested for 30 mins at 37°C in 1mg/ml collagenase I (Gibco, Life Technologies, NY, USA), 1mg/ml Dispase (Gibco, Life Technologies, NY, USA), 150μg/ml DNase-I (Sigma-Aldrich, St Louis, MO, USA) before passing the suspension through a 70μM cell strainer. Dissociated single cells in PBS/BSA/EDTA were then incubated with various antibody combinations for multi-parameter flow acquisition and analysis. The following combinations of antibodies were used to assess the endothelial hierarchy populations: Rat anti-mouse VEGFR2 PE, CD31 PE-Cy7 and CD34 Alexa647 (Becton Dickinson, NJ, USA), Rat anti-mouse Lineage cocktail BV450 (Biolegend), Rat anti-mouse CD144 FITC (eBioscience). FACS was performed on a FACSAria cell sorter (Becton Dickinson, Franklin Lakes, NJ, USA). scRNA-seq was performed in triplicate using wild-type C57/BL6 mice. The 10X Genomics Chromium instrument (10X Genomics) was used to partition viable cells with barcoded beads, and cDNA from each cell was prepared using the Single Cell 3' Library, Gel Bead and Multiplex Kit (v2; 10X Genomics) as per the manufacturer's instructions. Library construction performed as per 10X Genomic Single Cell 3' RNA-seq protocol version 2
attributes.tag: Experimental Factor: replicate
attributes.value: 2