EpiXplore Meth-Seq DNA Enrichment Kit
EpiXplore Meth-Seq DNA Enrichment Kit
Brand: Takara Bio.
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EpiXplore Meth-Seq DNA Enrichment Kit
EpiXplore Meth-Seq DNA Enrichment Kit
The EpiXplore Meth-Seq DNA Enrichment Kit is a complete system for separating methylated and unmethylated fractions of sheared genomic DNA and generating sequencing-ready Illumina libraries from the enriched DNA fractions. The enrichment protocol utilizes the his-tagged MBD2 protein currently used in the EpiXplore Methylated DNA Enrichment Kit in conjunction with Capturem Nanoprep Columns to separate methylated DNA from unmethylated DNA in ~2 hours. Illumina-compatible sequencing libraries are then generated from the methylated (or unmethylated, if desired) DNA fractions using DNA SMART technology.
For library prep, the DNA SMART method has been adapted for the addition of Illumina-specific sequencing adapters to the ends of DNA fragments in a ligation-independent manner. This highly sensitive technology generates robust sequencing libraries from low-input DNA samples, with minimal handling, in about four hours.
Overview
- Rapid, user-friendly protocol which separates methylated DNA from unmethylated DNA
- Ligation-independent sequencing-adapter addition for library preparation
- DNA inputs of 25 ng–1 μg sheared genomic DNA
- Generate Illumina-ready sequencing libraries from the enriched DNA in ~4 hours
Applications
- Sequencing library preparation for methylated DNA-seq from low-input, sheared genomic DNA
- Illumina-specific NGS sequencing library production
Flowchart for separating methylated and unmethylated fractions of DNA
Flowchart for separating methylated and unmethylated fractions of DNA. In the enrichment portion of the protocol, sheared DNA is incubated with his-tagged MBD2 protein for 1 hr, which allows the methylated DNA fraction to bind to the protein. The solution is then added to the Capturem nanoprep column, which binds the his-tagged protein and thus the methylated DNA. Subsequent washes with the Unmethylated Elution Buffer remove the unmethylated DNA. The methylated DNA is then eluted in a high-salt buffer. After cleanup, this enriched DNA can be used as the input for library preparation.
Flowchart for DNA-SMART ChIP-Seq technology
Flowchart for DNA-SMART ChIP-Seq technology. This single-tube workflow allows users to generate Illumina-compatible libraries for meth-seq experiments. After library size selection and purification, the total time from input DNA to meth-seq library is approximately four hours. DNA SMART technology eliminates the need for an adapter ligation step and associated cleanup, reducing the loss of limited input DNA.
Distinct methylation patterns are observed in aligned NGS data of libraries generated from methylated and unmethylated DNA fractions
Distinct methylation patterns are observed in aligned NGS data of libraries generated from methylated and unmethylated DNA fractions. A representative region of Arabidopsis chromosome 3 (outlined in the green box) shows the peaks identified in the DNA enriched for methylated and unmethylated fractions. The methylated regions are outlined in red boxes and the unmethylated regions are outlined in blue boxes. The amount of initial input DNA is shown along the left-hand side of the alignments. The data show good concordance with the publicly available bisulfite sequencing data. Data were visualized using the Integrative Genomics Viewer (Broad Institute). Public data was obtained from GEO accession GSM980986; Stroud et al., 2013.
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Flowchart for separating methylated and unmethylated fractions of DNA
Flowchart for separating methylated and unmethylated fractions of DNA. In the enrichment portion of the protocol, sheared DNA is incubated with his-tagged MBD2 protein for 1 hr, which allows the methylated DNA fraction to bind to the protein. The solution is then added to the Capturem nanoprep column, which binds the his-tagged protein and thus the methylated DNA. Subsequent washes with the Unmethylated Elution Buffer remove the unmethylated DNA. The methylated DNA is then eluted in a high-salt buffer. After cleanup, this enriched DNA can be used as the input for library preparation.
Flowchart for DNA-SMART ChIP-Seq technology
Flowchart for DNA-SMART ChIP-Seq technology. This single-tube workflow allows users to generate Illumina-compatible libraries for meth-seq experiments. After library size selection and purification, the total time from input DNA to meth-seq library is approximately four hours. DNA SMART technology eliminates the need for an adapter ligation step and associated cleanup, reducing the loss of limited input DNA.
Distinct methylation patterns are observed in aligned NGS data of libraries generated from methylated and unmethylated DNA fractions
Distinct methylation patterns are observed in aligned NGS data of libraries generated from methylated and unmethylated DNA fractions. A representative region of Arabidopsis chromosome 3 (outlined in the green box) shows the peaks identified in the DNA enriched for methylated and unmethylated fractions. The methylated regions are outlined in red boxes and the unmethylated regions are outlined in blue boxes. The amount of initial input DNA is shown along the left-hand side of the alignments. The data show good concordance with the publicly available bisulfite sequencing data. Data were visualized using the Integrative Genomics Viewer (Broad Institute). Public data was obtained from GEO accession GSM980986; Stroud et al., 2013.
