GSM8101971: Hepato_Male_NASH_1; Mus musculus; RNA-Seq
After cannulation of the inferior vena cava, the portal vein was cut, followed by retrograde stepwise perfusion with solutions containing protease (14 mg/mouse, Sigma Aldrich) and collagenase D (3.7 U/mouse, Roche). Livers were then minced with forceps. The minced livers were further digested in the solution containing 1% DNase, protease (25 mg/mouse) and collagenase D (4.4 U/mouse) for 25 min on a GentleMacs instrument. Digested livers were filtered through a 70-μm cell strainer to get rid of non-digested tissue. The cell suspensions were first centrifuged at 70 x g to isolate the primary hepatocytes which were stored in Qiazol at -80°C until sample processing. The supernatant was transferred to a fresh tube and again centrifuged at 580 x g for 10 min at 4°C. Cell pellets were washed with Gey's Balanced Salt Solution (Sigma Aldrich) twice. Cells were then subjected to gradient centrifugation on 9.7% Nycodenz (Accurate Chemical) to isolate hepatic stellate cells, which were then stored in Qiazol at -80°C until sample processing. RNA was isolated with RNeasy® Plus Micro Kit (Qiagen). Samples with a minimum RIN of 9.0 were prepared for sequencing using the NuGEN Ovation RNA-Seq system v2 reagents for cDNA preparation and the Ovation Ultralow DR Multiplex system for the adapter ligation step.