NextSeq 500 sequencing; RNA-seq of primary hepatocytes treated with GR and PPARalpha agonists in the presence of LPS
RNA-seq of primary hepatocytes treated with GR and PPARalpha agonists in the presence of LPS
Primary hepatocytes were isolated from 10–12 week-old male C57BL/6 mice by collagenase perfusion. RNA-seq was done in 3 biological replicates. Each replicate was obtained by pooling cells from 3–4 mice and then performing induction in 3 technical replicates. Isolated mRNA from these technical replicates was pooled to form 1 biological replicate. Hepatocytes were plated in the William's medium (Sigma, W1878), with 0,1% free-fatty acids and 1% glutamine in collagen-coated 6-well plates (BD Biosciences, cat. 356400) at the density of 0.75x106 cells. Cells were maintained in a 5% CO2 humidified atmosphere at 37°C. 19 h treatment with 1 uM Dexamethasone (GR ligand) and 0,5 uM GW7647 (PPAR alpha ligand) together with LPS (100 ng/ml 19 h)  RNA was isolated with the RNeasy purification kit (Qiagen, 74106) according to the user's manual. Library preparation and sequencing was done in VIB Nucleomics Core facility.