Genome binding/occupancy profiling by genome tiling array
Summary
The purpose of the chromatin immunoprecipitation/microarray (ChIP/chip) experiment is to determine which regions of a genome are enriched for a particular histone modification in a single Arabidopsis thaliana genotype. Chromatin immunoprecipitation with antibodies raised against dimethyl histone-H3 lysine-9 (H3mK9) or dimethyl histone-H3 lysine-4 (H3mK4) is performed on a selected genotype. This purified DNA from each immunoprecipiation (mH3K9, mH3K4, no antibody control) is used for random amplification to increase the quantity of DNA for microarray hybridization. The amplified DNA from each experimental sample is then labeled with Cy5 and hybridized against total input DNA from the corresponding genotype, labeled in Cy3. In a single hybridization, the total input DNA serves as a baseline and is compared to the immunoprecipitated samples. Ratios of normalized signal intensities were calculated to identify enrichment of a particular sequence after immunoprecipitation, in comparison to the total input DNA. Dye swap analysis is carried out to take account of experimental variation by repeating the hybridization with identical samples labeled with Cy3 and Cy5, respectively. The two samples in this series are complementary hybridizations in a dye-swap analysis These data were normalized and subjected to hypothesis testing. Error rate was controlled by Benjamini and Hochberg's step-up procedure for limiting the False Discovery Rate.
Ddm1 seedlings, 9 days old This is the normalized result of the paired dye swap samples EV59 and EV60. The ANOVA model of Kerr, Martin and Churchill (2000) was used to analyze the data from the dye-swap experiments, with terms included to account for gene, dye-by-gene, treatment-by-gene, and random error terms. The style of hypothesis test proposed by Black and Doerge (2002) was applied to each of the features represented on each array, with rejection of the null hypothesis indicating a significant change in fluorescence intensity. To account for the number of hypothesis tests being made, and thus provide some level of error rate control, significance was assessed using false discovery rate (FDR) controlling methods. The step-up procedure of Benjamini and Hochberg (1995) was used to control the FDR below alpha = 0.01. For the purposes of this experiment, the hypotheses were assumed to be independent. Features found after hypothesis-testing with a controlled error rate to be significantly enriched or depleted for H3K9 methylation compared to mean values found in euchromatic regions are flagged in the column. No family-wise error rate methods were used to analyze this sample. Keywords: other
