GSM5239501: D7_2; Mus musculus; RNA-Seq
For neonatal samples (D1, D3 and D7), decapitation was used for neonates sacrifice because they were resistance to hypoxia at this age. First, fetal livers were removed from body, cut up into small pieces and rinsed using Ca2+-free HBSS buffer (gibco) in order to reduce blood. Next, liver pellets were transferred into HBSS buffer with collagenase H (Roche) and DNase I (Roche), further dissociated with gentleMACS (Miltenyi) program m_liver_03 and then incubated at 37C for 30 minutes. After incubation, the additional gentleMACS program m_liver_04 was used for better dissociation result. Liver was passed through a 100um cell strainer. Cells were then centrifuged at 50g for 3 minutes, in order to separate hepatocytes (in pellets) from non-parenchymal cells (in suspension). In non-parenchymal cell suspension, ACK buffer (gibco) was added to lyse red blood cells. Hepatocytes and non-parenchymal cells were further washed with PBS separately, resuspended in DMEM with 10% FBS, counted with hemocytometer and mixed by ratio of cell numbers.  For D21 and D56 samples, 2 male mice were included in each time point. Mice were first sacrificed using CO2. Liver was perfused using two-step method with Ca2+-free HBSS buffer and then with collagenase H in HBSS buffer containing Ca2+, gently minced and passed through a 100um cell strainer. Cells were centrifuged at 50g for 3 min, in order to separate hepatocytes (in pallets) from non-parenchymal cells (in suspension). To remove dead cells and debris, the pelleted hepatocytes were resuspended in 45% Percoll (Cytiva) and centrifuged at 50g for 10 min, without brake. Meanwhile, the ACK buffer and Dead Cell Removal Kit were used to lyse red blood cells and eliminate dead cells in non-parenchymal cells. Hepatocytes and non-parenchymal cells were further washed with PBS separately, resuspended in DMEM with 10% FBS and counted with hemocytometer.  The isolated single cells were loaded onto 10x Chromium Controller. Chromium Single Cell 3' Reagent Kits (V2 chemistry, 10x Genomics)