Illumina HiSeq 4000 sequencing; Endothelial Cell Atlas
Endothelial Cell Atlas
Whole organs were dissected, followed by dissociation using tissue-specific digestion protocols. Samples were then enriched for endothelial cells (ECs) by magnetic bead sorting using the MACS system (Miltenyi Biotec). Next, positive selection of ECs was performed using CD31 MicroBeads (Miltenyi Biotec), followed by staining with various antibody combinations for multi-parameter flow acquisition. FACS-purification of ECs was then performed, and single cell suspensions of freshly isolated ECs were then resuspended in PBS containing 0.04% ultra-pure BSA. scRNA-seq libraries were prepared using the Chromium Single Cell 3′ Reagent Kits v2 (10x Genomics; Pleasanton, CA, USA) according to the manufacturer's instructions. The aimed target cell recovery for each library was 5000 (except for soleus, aimed recovery of 3000 cells). Generated libraries were sequenced on an Illumina HiSeq4000, followed by de-multiplexing and mapping to the mouse genome (build mm10) using CellRanger (10x Genomics, version 2.1.1). Samples were processed using V2 barcoding chemistry kits (10x Genomics). Single samples were always processed in a single well of a PCR plate, allowing all cells from a sample to be treated similarly. All samples were processed in parallel in the same thermal cycler.
Experimental Factor: organism part
spleen