Genomeplex wga2 kit

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.

ChIPs were performed as described [30 (link)]. In short, mouse tissues or mouse ES cells (CGR8; [56 (link)]) were fixed in 1% formaldehyde for 10 min after which nuclei were purified and sonicated using Bioruptor (Diagenode) to a length of 200 to 1000 bp. Samples were precleared with protein A+G-Sepharose (1:1) and immunoprecipitated with rabbit anti-Ring1b [57 (link)], rabbit anti-H3K27me3 (Upstate, 07–449), mouse anti-Ezh2 [58 ] overnight at 4°C. Immune complexes were collected by adsorption to protein A+G-Sepharose for 2 hr at 4°C. Beads were washed and immunocomplexes eluted prior to DNA purification with Qiaquick columns (Qiagen). Precipitations on pancreatic buds were performed with at least 20 pancreatic buds per IP and in the presence of 2.5 mg/mL BSA and 25 mg/mL tRNA. For tiling array experiments, ChIP and input DNA were amplified as described previously using the Sigma GenomePlex WGA2 kit while adding dUTPs to a final concentration of 0.4 mM during the amplification reaction to enable subsequent fragmentation [30 (link)]. We fragmented 6–7.5 μg DNA, labeled it using the Affymetrix GeneChip WT Double-Stranded DNA terminal Labeling Kit, and hybridized to GeneChip® Mouse Promoter 1.0R Arrays.