single cell RNA sequencing of liver cells in KLF9 and TSC22d1 knock-out mice
single cell RNA sequencing of liver cells in KLF9 and TSC22d1 knock-out mice
Liver cells were isolated using a modified protocol of Mederacke et al.22 In brief, using a peristaltic pump we performed retrograde liver perfusion into the inferior vena cava with three solutions: (1) EGTA (8 g/l NaCl, 0.4 g/l KCl, 88 mg/l NaH2PO4∙H2O, 120 mg/l Na2HPO4∙H2O, 2.38 g/l HEPES, 0.35 g/l NaHCO3, 0.19 g/l EGTA, 0.9 g/l glucose) for 2 min; (2) pronase (0.4 mg/ml Protease from Streptomyces griseus in EBS buffer: 8 g/l NaCl, 0.4 g/l KCl, 88 mg/l NaH2PO4∙H2O, 120 mg/l Na2HPO4∙H2O, 2.38 g/l HEPES, 0.35 g/l NaHCO3, 0.42 g/l CaCl2) for 5 min; and (3) collagenase D (0.1U/ml collagenase D in EBS buffer) for 7 min. The liver was then dissected, placed in cold EBS solution and shaken vigorously with forceps to allow separation of single cells. The solution was filtered through 100-μm mesh and hepatocytes were depleted by centrifugation at 30g for 5 min. Cells were then collected by centrifugation at 580g and resuspended in cold Ca/Mg-free PBS. To enrich for stellate cells, we sorted cells with retinoid fluorescence in the channel excitation 405, emission 450/40 using a BD FACSAria III. We then mixed stellate and unsorted cells in 1:1 ratio, spun them down, resuspended them in Ca/Mg-free PBS with 0.04% BSA and counted them using a Neubauer chamber, before proceeding to single-cell RNA-seq. according to 10x genomics Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 User Guide according to 10x genomics Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 User Guide