Illumina HiSeq 2500 sequencing; A Liver Hepcidin-Intestinal HIF-2α Axis Regulates Iron Absorption During Systemic Iron Deficiency, Hypoxia and Hemochromatosis
A Liver Hepcidin-Intestinal HIF-2α Axis Regulates Iron Absorption During Systemic Iron Deficiency, Hypoxia and Hemochromatosis
For temporal, hepatocyte-specific disruption of hepcidin, mouse floxed for Hamp1 (Hampfl/fl) on a C57BL/6 background were crossed with mice harboring CreERT2 recombinase under the control of the serum albumin promoter (AlbCreERT2) to generate AlbCreERT2;Hampfl/fl mice.  Wild-type littermates were used as controls for these respective experiments (Hampfl/fl). Mice were injected with tamoxifen (Sigma-Aldrich, St. Louis, MO) at 100 mg/kg of body weight via i.p. for three consecutive days to ensure Cre-mediated recombination. All mice were fed ad libitum and kept in a 12 hour dark/light cycle. All mice were fed with either a purified AIN-93G iron-replete (350 PPM) or low-iron (< 5 PPM) diet (Dyets, Bethlehem, PA) for two weeks prior to sacrifice. All mice were housed at the Unit for Laboratory Animal Management (ULAM) at the University of Michigan The small intestine tissue was scraped using a glass microscope slide and bead homogenized in Trizol (Thermo Fisher, Waltham, MA). RNA was extracted using chloroform (Sigma-Aldrich, St. Louis, MO) and isopropanol (Sigma-Aldrich, St. Louis, MO) RNA quality was assessed by the Agilent Bioanalyzer 2100. RNA sequencing libraries were prepared using the TruSeq RNA library prep kit v2 (Illumina, San Diego, CA) following the manufacturer's recommended protocol.
Experimental Factor: phenotype
hepatocyte-specific Hamp1 knockout
Experimental Factor: diet
low iron diet