DNase I Optimization for Whole Genome Amplification

DNase I treatment of cells was performed as previously described [8 (link)] with minor modifications. 1×10⁶ S2 cells were permeabilized with 0.05% NP40 and resuspended in DNase I Buffer (40 mM Tris–HCl, 0.4 mM EDTA, 10 mM MgCl2, 10 mM CaCl2, 0.1 mg/ml BSA). Part of each sample was set aside and later used as non-digested control. DNase I (Promega) digestion was performed at 37°C for 10 or 15 minutes with diverse amounts of the enzyme to optimize the procedure (0.1U, 0.5U, 1U, 2U and 5U); for further analysis cells were treated with 0.5U DNase I for 10 minutes. 20 ng of DNA purified from the treated cells using the DNeasy Blood & Tissue Kit (Qiagen) were used as a template for whole genome amplification. The library preparation step using GenomePlex WGA2 Kit was followed by amplification with the GenomePlex WGA 3 Reamplification Kit (Sigma). dUTP was incorporated at the amplification step to enable the probe fragmentation procedure according to Affymetrix recommendations. The amplification product was purified with the Wizard SV Gel and PCR Clean-Up System (Promega), fragmented and labeled using the GeneChip WT Double-Stranded DNA Labeling Kit (Affymetrix), and hybridized with GeneChip Drosophila Tiling 2.0R Array following the manufacturer’s instructions.