DNA methylation in Arabidopsis thaliana

DNA methylation in wild type bolting plants, wild type seedlings, and ddm1 seedlings. The purpose of the McrBC methylation microarray assay is to determine which regions of a genome are methylated versus those that are unmethylated in a single Arabidopsis thanliana genotype. McrBC is a methylation-sensitive enzyme that restricts DNA only at purine-Cmethyl half sites when separated between 50bp and 3kb. A designated amount of DNA from a particular genotype is sheared to a size range of 1kb-10kb using nebulization. We restrict half of the nebulized DNA with McrBC, and the methylated fraction is then removed from the unmethylated fraction through gel purification of DNA fragments greater than 1kb.* The remaining nebulized DNA is subjected to the same gel purification scheme, but with no McrBC treatment. In a single hybridization, the untreated sample is labeled with Cy5 and the McrBC-treated sample with Cy3. Thus, after labeling and microarray hybridization, the ratio of normalized Cy5 to normalized Cy3 represents the relative methylation at the sequence represented by the spot on the microarray. 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. This SuperSeries is composed of the SubSeries listed below. Overall design: Refer to individual Series

本数据集针对野生型抽薹植株、野生型幼苗及ddm1幼苗开展DNA甲基化分析。本研究采用McrBC甲基化微阵列检测技术（McrBC methylation microarray assay），旨在明确单一拟南芥（Arabidopsis thaliana）基因型的基因组中，甲基化区域与非甲基化区域的分布情况。McrBC是一种甲基化敏感型限制性内切酶，仅在间距为50bp至3kb的嘌呤-C甲基化半位点处切割DNA。取特定基因型的定量DNA，通过雾化法（nebulization）将其片段化至1kb~10kb的长度范围。我们将一半经雾化处理的DNA用McrBC进行酶切，随后通过凝胶纯化回收大于1kb的DNA片段，以此分离甲基化组分与未甲基化组分。剩余的另一半雾化DNA则采用相同的凝胶纯化流程，但不经过McrBC酶切处理。在单次杂交实验中，未酶切的样品以Cy5标记，经McrBC酶切的样品以Cy3标记。完成标记与微阵列杂交后，标准化后的Cy5与Cy3信号强度比值，即可反映微阵列探针对应序列位点的相对甲基化水平。为抵消实验变异带来的影响，我们开展了荧光互换（dye swap）分析：将相同样品分别以Cy3和Cy5标记后重复杂交实验。本超级数据集（SuperSeries）由以下所列的子数据集（SubSeries）组成。整体实验设计详见各子数据集说明。