The EpiQuik™ Chromatin Accessibility and Methylome Sequencing (CAMe-Seq) Kit is a complete set of optimized reagents designed for conducting a gene-specific or genome-wide analysis of both chromatin accessibility and DNA methylation simultaneously from various biological samples at a single molecule or population level, followed by bisulfite-sequencing or next generation sequencing. The kit has the following advantages and features:
- Extremely fast and convenient protocol that allows the entire procedure (from cell tissue sample to ready-to-use DNA for cloning/library preparation) to be finished in as short as 5 hours.
- Simultaneously detect chromatin accessibility (nucleosome/transcription factor positioning) and CpG methylation at a base-resolution level.
- Internal control primers are included in the kit for validating whether the proper enzymatic DNA methylation and bisulfite modification are achieved.
- Recommended procedures for cloning and sequencing are included.
The accessibility of regulatory elements in chromatin is critical for many aspects of gene regulation. Nucleosomes positioned over regulatory elements inhibit access of transcription factors to DNA. To elucidate the role of the interactions between chromatin and transcription factors, it is crucial to determine chromatin accessibility through mapping of the nucleosome positioning along the genome. DNA methylation occurs by the covalent addition of a methyl group at the 5-carbon of the cytosine ring, resulting in 5-methylcytosine. When a CpG island in the promoter region of a gene is methylated, expression of the gene is repressed. It has been demonstrated that nucleosome positioning influences DNA methylation patterning throughout the genome. Thus, obtaining DNA methylation and nucleosome positioning information from the same DNA molecule would enable us to better understand how DNA methylation and nucleosome positioning work together to control chromatin accessibility that regulates gene expression.
Principle & Procedure
The CAMe-Seq Kit contains all the necessary reagents required for the assay. In CAMe-Seq, the cells/tissues are fixed with formaldehyde. Chromatin is isolated and treated with the included GpC DNA methyltransferase that only methylates GpC sites unprotected by nucleosomes or DNA/protein complexes without affecting endogenous CpG sites. DNA is then bisulfite modified after reversal of cross-linking. The bisulfite-converted DNA can be PCR amplified with or without cloning followed by sequencing for region-specific analysis of chromatin accessibility and DNA methylation. The C to T conversion in the GpC sites indicates accessible euchromatin regions while unchanged GpC sites represent chromatin regions that are not accessible because of the presence of nucleosome or DNA/protein complexes. The GpC methylated and bisulfite-converted DNA obtained with this kit can also be used in next generation sequencing to analyze genome-wide chromatin accessibility profiles and DNA methylation footprints.
Starting Materials & Input Amount
Starting materials can include various mammalian tissue or cell samples such as cells from flask or microplate cultured cells, fresh and frozen tissues, etc.The amount of cell/tissues for each reaction can be from 1 x 105 cells or 2 mg tissues to 1 x 106 cells or 20 mg tissues. For an optimal reaction, the input chromatin amount should be about 0.5 x 106 cells or 10 mg tissues.
Fig. 1. Schematic procedure of the EpiQuik™ Chromatin Accessibility and Methylome Sequencing (CAMe-Seq) Kit.
Fig. 2. qPCR amplification of a gene region in euchromatin with internal control primers. Successful GpC methylation and bisulfite conversion using the P-1048 kit give a CT Value of 5 to 8 cycles less for Mse-treated samples than untreated samples. Green/Red: Mse-treated; Blue: Mse-untreated
Fig. 3. DNA sequencing results after GpC methylation and bisulfite conversion. DNA was GpC methylated followed by bisulfite treatment by using the P-1048 kit. The recovered DNA was amplified by PCR with the internal control primers and then sequenced directly.