Title: Affymetrix CEL analysis. Description:
Affymetrix Generic Hybridization
DNA of sizes above 17 kb was amplified and labelled using the BioPrime(r) DNA Labelling System (Invitrogen, 18094-011, CA).
Crude nuclei extracts were produced by treating 100 mg of ground frozen plant material in Modified Nuclei Extraction Buffer (MEB; 20 mM PIPES-KOH pH 7.6, 1 M hexylene glycol, 10 mM MgCl2, 1 mM EGTA, 15 mM NaCl, 0.5 mM spermidine, 0.15 mM spermine, 0.5% Triton-X100, 5 mM beta-mercaptoethanol and EDTA-free protease inhibitor cocktail (Roche, Switzerland)) for 15 min at 4degrees C. The homogenate was filtered through Miracloth (Calbiochem, Germany), and a pellet was collected by a 5 min centrifugation at 1500 rcf at 4 degrees C. Isolated nuclei were washed once in Nuclei Washing Buffer (NWB; 40 mM Tris-HCl pH 8 0.3 M sucrose, 10 mM MgSO4, and EDTA-free protease inhibitor cocktail (Roche)) and were used for DNase I (Promega, Wisconsin) digestion for 15 min (final concentration 4.5 U/ml for treatment and 0 U/ml for background control samples) at 30degrees C in Digestion Buffer (NWB plus 1 mM CaCl2). The reaction was stopped with 50 mM EDTA. The digested nuclei mixture was deproteinized using Proteinase K (Sigma-Aldrich, Missouri) and DNA was extracted using careful phenol-chloroform extraction and ethanol/salt precipitation. Recovered DNA was redissolved and resolved on agarose gels. Gel slabs containing DNA fragments of sizes above 17 kb were excised, and DNA was extracted using the freeze/thaw method. DNA of sizes above 17 kb was amplified and labelled using the BioPrime(r) DNA Labelling System (Invitrogen, California).