Single cell RNA-seq of intrahepatic biliary epithelial cells following bile duct ligation.

To identify biomarkers of subpopulations of intrahepatic biliary epithelial cells, we performed single cell RNA-seq on adult biliary epithelial cells (BECs) and hybrid hepatocytes (HybHeps) from uninjured or cholestatic injured Sox9EGFP+ mice. After 7 days, the livers were dissociated to single cells and stained for CD31, CD45, Annexin V, and 7ADD. BECs were isolated by first excluding positively stained cells and then sorting EGFP+ cells. Gallbladder and extrahepatic tissues were excluded by dissection. Sorted cells were fixed according to manufacturer instructions using Parse Whole Cell Fixation kit. Fixed cells were frozen and stored at -70C for less than 6mo, prior to being used in split-pool combinatorial barcoding according to manufacturer instructions. Barcoding and library construction was carried out with the Parse Evercode Whole Transcriptome v2 kit. Overall design: Clustering and differential expression analysis identified 9 BEC and 6 non-BEC clusters.