normalization data transformation protocol
Selection of probes matching the D. rerio genome (bitscore 119), and not matching the genome (bitscore < 40). ID_REF = VALUE = Log2-transformed raw expression value
array scanning protocol
The slides were washed according to the NimbleGen Arrays User's Guide (Gene Expression Arrays Version 5.0) and scanned in an ozone-free room with an Agilent DNA microarray scanner G2565CA.
Each hybridization mixture was made up from 1 µg test material (Cy3) and 1 µg reference material (Cy5). The D. rerio experiments were performed according to a common reference design, with twelve individuals as test samples and a pool as common reference. Details for the D. rerio microarray can be found in Rauwerda et al., 2010 (PMID 20626891). It is a three prime biased expression microarray with 63,751 target probes and 7,742 probes with random sequences not targeting to the Danio rerio genome, which we used as negative-control probes.
Genomic DNA was amplified and labeled by strand displacement amplification. 200 ng genomic DNA was combined with 5 µg random octamers (Biolegio). The DNA was partly fragmented by heat at 99°C for 10 min. and allowed to cool on an ice/water bath for 5 min. The DNA/octamer mix was made on ice to 50 mM Tris-Cl (pH 7.5), 5 mM MgCl2, 1 mM DTT, 1 mM dGAC (GE Healthcare), 0.6 mM dTTP (GE Healthcare), 0.4 mM aminoallyl dUTP (TriLink Biotechnologies) and finally 10U Klenow exo- (Jena Bioscience) was added. After gentle vortexing, the samples were placed at 37°C for 8h. and subsequently incubated for 15 min. at 75°C to inactivate the enzyme. Each reaction was made to 1.5M sodium acetate (pH 5.2) and the amplified DNA was purified with the QIAquick PCR Purification Kit (Qiagen) according to the manufacturer's instructions with the exception that 7 volumes of buffer PB were used instead of 5 to increase the recovery of smaller fragments. Concentrations of amplified products were measured on the NanoDrop ND-1000 (Thermo Scientific) and quality assessed on the BioAnalyzer (Agilent Technologies) with the DNA 1000 Kit (Agilent Technologies). From each sample, 5 µg was taken, dried down and dissolved in 50 mM carbonate buffer (pH 8.5). Individual vials of Cy3/Cy5 from the mono-reactive dye packs (GE Healthcare) were dissolved in 200 µl DMSO. To each sample, 10 µl of the appropriate CyDye dissolved in DMSO was added and the mixture was incubated for 1h. Reactions were quenched with the addition of 5 µl 4M hydroxylamine (Sigma-Aldrich). This mixture was made to 0.75M Ammonium acetate and 0.2 µg/µl glycogen (Ambion). After mixing, 2.5 vol. of 100% EtOH was added and mixed. All samples were incubated for at least 60 min. at -20°C to enhance precipitation and centrifuged for 30 min. at 13.000xg. The supernatants were carefully removed by pipetting and 1 vol. of 80% EtOH was added to the pellets. After brief vortexing, the labeled DNA was pelleted once more by centrifugation for 10 min. at 13.000xg. The supernatants were removed by pipetting and the pellets were dried by vacuum concentrating. Final labeled pellets were dissolved in 30 µl H2O, and the yield and CyDye incorporation were measured with the NanoDrop ND-1000.
nucleic acid extraction protocol
Genomic DNA was extracted using a CTAB DNA extraction method that included an RNAse A (Sigma-Aldrich) digestion step for removal of residual RNA.