Male C57BL/6N mice (10 weeks old, 25-28 g) from Janvier (France) had free access to standard rodent chow and drinking water. Up to 6 mice were housed per cage in 12-h day and night cycles.
Extracted molecule
total RNA
Extraction protocol
24 h after glycerol injection, animals were euthanized by cervical dislocation under isoflurane narcosis, and kidneys were rapidly removed, halved, and snap frozen in liquid nitrogen. Halved mouse kidneys were ground into fine powder in liquid nitrogen. RNA was isolated using RNA-Bee (AMS Biotechnology (Europe) Ltd - Germany) according to the manufacturer’s instructions.
Label
biotin
Label protocol
RNA integrity was checked by analyzing on the 2100 Bioanalyzer (Agilent technologies, PA, USA). For the first and subsequent second strand cDNA synthesis 200 ng of total RNA was used and, also, poly-A Spike Controls (containing several B. subtilis genes, that are absent in eukaryotic samples) are added in the recommended dilution to achieve the final concentrations corresponding to the given RNA amount of 200 ng. During the following in vitro transcription reaction cRNA was obtained and used as starting material for the second cycle cDNA synthesis. The second cycle random-primed single strand DNA synthesis resulted in a product containing incorporated deoxyuridine at predefined ratios relative to thymidine, according to the manufacturers recommendation: WT Expression Plus Kit (Affymetrix Inc., Santa Clara, CA, USA). Subsequently, 5.5 μg of this generated single-strand DNA was fragmented with a combination of uracil DNA glucosylase (UDG) and apurinic/apyrimidinic endonuclease 1 (APE1) and then labeled. The biotinylated DNA was added to a hybridization cocktail for hybridization to the Mouse Clariom S Array, also containing 50 pM control oligonucleotide B2, eukaryotic hybridization controls (bioB, bioC, bioD, 1,5, 5; 25 pM respectively), accordingly to the manufacturer’s protocol (Hybridization Wash and Stain Kit, Affymetrix).
Hybridization protocol
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.
Scan protocol
GeneChipScanner 3000 G7 system (as recommended by Affymetrix (Affymetrix Inc., Santa Clara, CA, USA)).
Description
control animals & control molecules
Data processing
Data were processed using Partek Genomics Suite 6.4 software and normalization according to RMA method. Array control features have been filtered out.
