RNA-sequencing in human primary hepatocytes lacking NREP

To evaluate the global transcriptomic changes induced by Neuronal Regeneration Related Protein (NREP) downregulation, we employed RNA-sequencing in human primary hepatocytes lacking NREP. We used pure, highly viable, and plateable primary human hepatocytes from 5 pooled donors (ThermoFisher, #HMCPP5). Briefly, hepatocytes were seeded on 6-well collagen-treated plates containing William E media supplemented with primary hepatocyte supplements (ThermoFisher) and HepExtend supplement (ThermoFisher). Hepatocytes were allowed to attach for 6h. Media was exchanged to eliminate non-viable/attached cells that were forward transfected with Lipofectamine RNAiMAX (ThermoFisher) and 100nM of siGENOME Non-Targeting siRNA Pool (Dharmacon, # D-001206-13-05), or siGENOME human NREP siRNA (Dharmacon, # M-019848-00-0005) in complete media. Media was then changed at 6h post-transfection with complete media. At 48h post-transfection cells were starved for 16h in William E media containing 0.1% BSA, washed three times with ice-cold DPBS, and snap-frozen and pelleted for further analyses.