GSM3728306: LEC-enriched hepatic non-parenchymal cells (NASH); Homo sapiens; RNA-Seq
To enrich LECs from hepatic NPCs we thawed frozen samples in RPMI containing 10% Human Serum AB (Gemini Bio-products, West Sacramento, CA) and 1% DNASE (MP Biomedicals, Santa Ana, CA). Cells were washed 2x with PBS containing 2% FBS (Atlas Biologicals, Fort Collins, CO) and stained with antibodies against CD45 (clone HI30), CD31 (PECAM1, clone WM59), Podoplanin (clone NC-08) and CD146 (clone P1H12) from Biolegend (San Diego, CA) and CD68 (clone KP1) from abcam (San Francisco, CA). Cells from either Non-diseased, NASH or HCV were sorted using an Aria Fusion sorter (BD Biosciences, Franklin lakes, NJ) and enriched LECs were sorted into RPMI containing 50% human serum AB. Approximately 10,000 LEC-enriched hepatic NPCs were loaded onto a 10x genomics (San Francisco, CA) controller per manufacturer instructions to generate barcoded single cell GEMs using the 10x genomics 3' kit. 10x Genomics 3' end library kit