Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution
| Autor: | Thadeous J. Kacmarczyk, Francine E. Garrett-Bakelman, Alicia Alonso, Ari Melnick, Doron Betel, Jennifer Ishii, Maria E. Figueroa, Christopher E. Mason, Caroline Sheridan |
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| Jazyk: | angličtina |
| Rok vydání: | 2015 |
| Předmět: |
genomic DNA
General Chemical Engineering Bisulfite sequencing Molecular Sequence Data Biology DNA sequencing General Biochemistry Genetics and Molecular Biology 03 medical and health sciences chemistry.chemical_compound Cytosine 0302 clinical medicine Genetics Humans Sulfites Methylated DNA immunoprecipitation 5-methylcytosine Issue 96 Base Pairing high-throughput 030304 developmental biology 0303 health sciences DNA methylation General Immunology and Microbiology Base Sequence General Neuroscience DNA Restriction Enzymes Sequence Analysis DNA 5-Methylcytosine CpG site chemistry 030220 oncology & carcinogenesis Reduced representation bisulfite sequencing Illumina Methylation Assay bisulfite sequencing Epigenetics CpG Islands |
| Zdroj: | Journal of Visualized Experiments : JoVE |
| ISSN: | 1940-087X |
| Popis: | DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine methylation patterns in cells. Reduced representation of whole genome bisulfite sequencing was developed to detect quantitative base pair resolution cytosine methylation patterns at GC-rich genomic loci. This is accomplished by combining the use of a restriction enzyme followed by bisulfite conversion. Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) increases the biologically relevant genomic loci covered and has been used to profile cytosine methylation in DNA from human, mouse and other organisms. ERRBS initiates with restriction enzyme digestion of DNA to generate low molecular weight fragments for use in library preparation. These fragments are subjected to standard library construction for next generation sequencing. Bisulfite conversion of unmethylated cytosines prior to the final amplification step allows for quantitative base resolution of cytosine methylation levels in covered genomic loci. The protocol can be completed within four days. Despite low complexity in the first three bases sequenced, ERRBS libraries yield high quality data when using a designated sequencing control lane. Mapping and bioinformatics analysis is then performed and yields data that can be easily integrated with a variety of genome-wide platforms. ERRBS can utilize small input material quantities making it feasible to process human clinical samples and applicable in a range of research applications. The video produced demonstrates critical steps of the ERRBS protocol. |
| Databáze: | OpenAIRE |
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