Name: GSM6472997
Instrument: Illumina HiSeq 4000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: Mice were anesthetized by intraperitoneal injection of sodium pentobarbital (Nembutal, 50mg/kg). Livers were perfused for 5min with 40ml of perfusion medium SC-1 to remove the blood and for rapid destruction of intercellular junctions, followed by perfusion with 40ml of SC-2 medium containing 10mg collagenase for 5min to break up the supporting extracellular matrix. Each lobe was dissected off, and the tissue was disrupted inside of a beaker containing 50ml of SC-2 with 20mg collagenase (Roche) and 1ml DNase I (Sigma) followed by rotating incubation for 20min at 37°C. Cells were then filtered through a 100μm strainer and centrifuged at 50g for 2min at room temperature. Hepatocytes were washed once with 1X PBS and centrifuged again at 50g for 2min. Pellet was resuspend with 2ml of 1X PBS containing 0.04% BSA, filtered through a 70µm strainer and FACS sorted. 500,000 sorted cells were collected and lysed for RNA extraction. RNA was extracted immediately using the RNAeasy mini spin kit (Qiagen) and stored at -80°C. Library preparation was performed according to the QuantSeq 3' mRNA-Seq Library Prep kit FWD (Lexogen).