RNA-sequencing in human primary hepatocytes lacking NREP. 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. Overall design: 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.

为评估神经元再生相关蛋白（Neuronal Regeneration Related Protein, NREP）下调所诱导的全局转录组变化，我们在NREP下调的人原代肝细胞中开展了RNA测序（RNA-sequencing）实验。
总体实验设计：我们采用了来自5名混合供体的高纯度、高活性且可贴壁的人原代肝细胞（ThermoFisher，货号#HMCPP5）。具体操作步骤如下：将肝细胞接种于经胶原蛋白包被的6孔板中，培养基为添加了原代肝细胞添加剂（ThermoFisher）与HepExtend添加剂（ThermoFisher）的William E培养基。待肝细胞贴壁6小时后，更换培养基以清除未存活或未贴壁的细胞；随后使用Lipofectamine RNAiMAX（ThermoFisher）转染试剂，将100nM的siGENOME非靶向siRNA池（Dharmacon，货号# D-001206-13-05）或siGENOME人源NREP siRNA（Dharmacon，货号# M-019848-00-0005）加入完全培养基中进行转染。转染后6小时更换一次完全培养基。转染后48小时，将细胞置于含0.1%牛血清白蛋白（BSA）的William E培养基中饥饿处理16小时，随后用预冷的DPBS洗涤三次，将细胞速冻并离心收集沉淀以用于后续分析。