At light onset, animals were either sleep deprived by gentle handling or left undisturbed for 6 hrs.
Experiments were performed on 12 weeks-old (+/- 1 week) male mice. Animals were sleep deprived at light onset or left undisturbed for 6 hrs and randomly sacrificed by cervical dislocation.
Mice were sacrificed by cervical dislocation. Total RNA: Whole brain total RNA was extracted and treated with DNase using the RNeasy Lipid Tissue Kit (Qiagen). PolyA RNA: the mRNA-tagging immunoprecipitation was modified from formerly published protocols. (P. J. Roy et al., 2002) (L. O. F. Penalva et al., 2004). Mice were anesthetized with pentobarbital (75mg/kg), and subsequently perfused with 20ml PBS-heparin (2U/ml), 20 ml PBS-formaldehyde 1%, 15ml gylcine 150mM, 20ml Tris-HCl 20mM, and 15ml NaCl 150mM. Brains were removed and homogenized at maximum speed on ice in 3ml lysis buffer (Hepes KOH pH7.3 50mM, EDTA 1mM, KCl 140mM, glycerol 10%, NP40 0.5%, dithiothreitol 1mM, vanadyl ribonucleoside complex 2mM, heparin 1000U/ml, 1 complete mini protease inhibitor cocktail tablet/50ml). Large debris were removed by centrifugation at 18’000g for 20 minutes at 4°C. The supernatants were incubated with 400μl of anti-FLAG M2 affinity gel beads (Sigma) and 80U Rnasine overnight at 4°C. After centrifugation, the pull-down supernatants were recovered for total RNA extraction. The beads were then washed three times with 7ml of lysis buffer, twice with 7ml of wash buffer (50mM Hepes-KOH pH7.3, 1mM EDTA, 1M KCl, 10% glycerol, 0.5%NP-40, 1mM DTT) and once with 7ml of TE (10mM Tris-HCl pH 7.5, 0.5mM EDTA). The RNA-protein crosslink was reversed by incubating the beads with 3ml of elution buffer for 6 hours at 65°C (50mM Tris-HCl pH7.5, 10mM EDTA, 1% SDS, 2mM ribonucleoside vanadyl complex). Proteins were then digested with proteinase K for 45 minutes at 50°C. Finally mRNAs were isolated from proteins and DNA by a double phenol-chloroform extraction followed by isopropanol precipitation. The mRNA pellets were rinsed twice with 0.5ml 70% ethanol and dissolved in 20μl RNase free water.
The quality of the mRNAs was investigated on Agilent 2100 bioanalyzer chips. 10ng of immunoprecipitated mRNA was amplified with the WT-Ovation™ Pico RNA Amplification System (catalog #3300-12; Manuel version 1,0).Using the FL-Ovation cDNA Biotin Module V2 (catalog #4200-12) 5μg of cDNA was used to generate labeled targets for hybridization to Affymetrix GeneChip® arrays.
Affymetrix HG-U133 plus 2.0 microarrays (Affymetrix) were hybridized to 5 ug of labeled, amplified material and then, washed, stained and scanned according to the protocol described in Affymetrix GeneChip® Expression analysis manual. The fluidics protocol used was GE_WS2v4_450 as recommended by NuGen and not the GE_WS2v5_450 used for the Affymetrix amplification.
The GeneChip Scanner 3000 (Affymetrix) was used to collect signals after excitation at 570 nm.
Normalized expression signals were calculated from Affymetrix CEL files using the expresso function from Bioconductor affy package. This normalization follows the same procedure as for RMA, but replaces a quantiles normalization by a scaling normalization (Gautier et al.).