normalization data transformation protocol
Data were preprocessed and normalized using the affy package in R. ID_REF = VALUE = RMA-normalized intensity
array scanning protocol
GeneChips were scanned using the GeneChip Scanner 3000 G7 system as recommended by Affymetrix.
The biotinylated DNA was added to a hybridization cocktail for hybridization to the Affymetrix Mouse Gene 1.0 ST Array, also containing 50pM control oligonucleotide B2, eukaryotic hybridization controls (bioB, bioC, bioD, 1,5, 5; 25 pM respectively), according to the manufacturer’s protocol (Hybridization Wash and Stain Kit, Affymetrix). The hybridization in the Affymetrix hybridization oven 640 at 45°C and 60 rpm for 16hrs, the staining and washing, processed in the Fluidics Station 450.
For the first cycle cDNA synthesis, 300ng of total RNA was used. 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 (Ambion® WT Expression Kit (Ambion, Austin, Texas, 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 using the WT terminal labeling kit (Affymetrix, Inc., Santa Clara, CA, USA).
nucleic acid extraction protocol
Trizol extraction of total RNA was performed according to the manufacturer's instructions. RNA integrity was checked by analyzing on the 2100 Bioanalyzer (Agilent Technologies, PA, USA).
sample treatment protocol
Subsequently, animals were transferred into constant darkness (DD) and killed by cervical dislocation at 4 h intervals starting 24 hours after lights off (= circadian time (CT) 12). The bilateral OEs were then explanted and shock-frozen in liquid nitrogen.
48 male C57BL/6 mice were exposed to a 12 h : 12 h light/dark (LD) cycle for at least 14 days to ensure proper entrainment of the circadian system to the ambient LD cycle.