normalization data transformation protocol
Data were processed using Partek Genomics Suite 6.4 software. Normalization according to RMA method. MoGene-1_0-st-v1 ID_REF = VALUE = log2 RMA signal
array scanning protocol
Affymetrix Gene ChIP Scanner 3000 7G
The biotinylated cDNA was added to a hybridization cocktail for hybridization to the Mouse Gene 1.0ST Array (Hybridization Wash and Stain Kit, Affymetrix). The hybridization in the Affymetrix hybridization oven 640 at 45°C and 60 rpm for 16 h, the staining and washing processed in the Fluidics Station 450.
Random-primed cDNA synthesis was performed using the Ambion® WT Expression Kit (Ambion, Austin, Texas, USA). Subsequently, cDNA was fragmented with a combination of uracil DNA glucosylase (UDG) and apurinic/apyrimidinic endonuclease 1 (APE1) and then labeled using the WT terminal labeling kit (Affymetrix Inc., Santa Clara, CA, USA).
sample treatment protocol
Doxycycline (0.1 mg per 10 g body weight SC) was injected 4 days prior to sacrifice (Groups: VHL-KO and VHL-KO/AKI). To induce AKI, 50% glycerol (0.05 ml per 10 g body weight) was injected IM into the left hind limb under isoflurane narcosis (Groups: AKI AND VHL-KO/AKI). Drinking water was withdrawn between 20 h prior and 24 h after glycerol injection.
nucleic acid extraction protocol
For each condition, kidneys were harvested after cervical dislocation under isoflurane anaesthesia, and processed for homogenization. Tissue homogenates were performed from transversally sectioned fresh kidney halves which were snap frozen in liquid nitrogen immediately thereafter. Total RNA from halved kidneys was isolated using RNA-Bee (AMS Biotechnology (Europe) Ltd - Germany) according to the manufacturer’s instructions. RNA integrity was checked by analyzing on the 2100 Bioanalyzer (Agilent technologies, PA, USA).
Mice of both sexes (24 to 31 g body weight) were fed a standard rodent chow with 19% protein content (V1534/300, Ssniff, Soest, Germany), and had free access to drinking water unless otherwise specified. Up to 5 mice were housed per cage in 12 h day and night cycles. All experiments were carried out in transgenic mice in which selective renal tubular VHL knockout (VHL-KO) was inducible by doxycycline.