GSM7767407: caMEK1_GFP; Mus musculus; RNA-Seq
Hepatocytes were isolated by a modification of a previously described two-step collagenase perfusion method. Mice were anesthetized, and inferior vena cava was canulated under aseptic conditions. The liver was perfused EGTA solution (calcium-free Hank's Balanced Salt Solution (HBSS) (Wako) containing 0.5mM EGTA (Sigma) and 10mM HEPES (Nacalai Teque)), followed by a second perfusion with HBSS containing Liberase TM Research Grade (Roche) and 10mM HEPES. After passage through 70µm cell strainer, to enrich hepatocytes, the cells were centrifuged at 50 g for 2 minutes three times. The viability of cells was confirmed by trypan blue staining. Dissociated hepatocytes were passed through 40 µm cell strainer and sorted with FACSAriaⅡ (BD Bioscience) machine using a 130 µm nozzle. Dead cells were gated out by DAPI incorporation (500 ng/mL). To enrich hepatocytes, we used 2 FSC-neutral density (ND) filter and 10-SSC ND filter. Cells were sorted into low glucose DMEM (Nacalai Teque) with 10% fetal bovine serum (Gibco) and 10 mM HEPES and proceeded to the 10X pipeline. Cells isolated from three mice were loaded into a 10X Genomics Chromium system. scRNA-seq libraries were prepared using Single Cell 3' Reagent Kits (v.3.1, 10X Genomics).