GSM6632504: liver IDD_HID scRNAseq replicate 1; Mus musculus; RNA-Seq
Anaesthetized mice were perfused as previously described4 with a few modifications5. Calcium-free Hank's Balanced Salt Solution (HBSS) buffer containing 0.2 mg/ml EDTA was used to perfuse the liver followed by sequential perfusion with calcium-containing HBSS and 0.5 mg/ml Collagenase D (Roche) for 7 minutes. The liver was excised and minced in 5 ml warm perfusion buffer before addition of 0.5 mg/ml pronase (Sigma; P5147) and 0.1 mg/ml DNase I (Roche) in a total volume of 50 ml. Ex vivo digestion was performed at 37oC for 20 min before filtering through a 70 μM filter. Cells were pelleted and collected by spinning at 580 g/10 min/4℃ and resuspended in 25 ml DMEM. A series of 4 spins at 50 g/2 min/4℃ were performed to pellet hepatocytes and collect non-parenchymal cells (NPCs) in supernatant. NPCs were then pelleted at 580 g/10 min/4℃ and treated with RBC lysis buffer (eBioscience™) according to the manufacturer's instructions before resuspension in 5 ml sequencing buffer (phosphate-buffered saline containing 0.04% bovine serum albumin). Hepatocyte viability selection was performed as described6 on the hepatocyte pellet and cells were resuspended in sequencing buffer. A final mixture of cells was made by mixing 5% of the hepatocyte suspension with 95% of the NPC suspension before adjusting the cell concentration to 4000 cells/μl. Single-cell suspensions were processed on the 10X scRNA-Seq platform (Chromium Next GEM Single Cell 3' GEM Kit v3.1)