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DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments.
- Source :
-
Journal of visualized experiments : JoVE [J Vis Exp] 2016 Jan 27 (107), pp. e53620. Date of Electronic Publication: 2016 Jan 27. - Publication Year :
- 2016
-
Abstract
- The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is an alternative approach to chromatin immunoprecipitation (ChIP). An expressed fusion protein consisting of the protein of interest and the E. coli DNA adenine methyltransferase can methylate the adenine base in GATC motifs near the sites of protein-DNA interactions. Adenine-methylated DNA fragments can then be specifically amplified and detected. The original DamID assay detects the genomic locations of methylated DNA fragments by hybridization to DNA microarrays, which is limited by the availability of microarrays and the density of predetermined probes. In this paper, we report the detailed protocol of integrating high throughput DNA sequencing into DamID (DamID-seq). The large number of short reads generated from DamID-seq enables detecting and localizing protein-DNA interactions genome-wide with high precision and sensitivity. We have used the DamID-seq assay to study genome-nuclear lamina (NL) interactions in mammalian cells, and have noticed that DamID-seq provides a high resolution and a wide dynamic range in detecting genome-NL interactions. The DamID-seq approach enables probing NL associations within gene structures and allows comparing genome-NL interaction maps with other functional genomic data, such as ChIP-seq and RNA-seq.
- Subjects :
- 3T3 Cells
Adenine metabolism
Animals
DNA genetics
DNA metabolism
Genomics methods
Humans
Mice
Oligonucleotide Array Sequence Analysis methods
Sequence Analysis, DNA methods
Site-Specific DNA-Methyltransferase (Adenine-Specific) genetics
Site-Specific DNA-Methyltransferase (Adenine-Specific) metabolism
Chromosome Mapping methods
DNA chemistry
DNA Methylation
High-Throughput Nucleotide Sequencing methods
Site-Specific DNA-Methyltransferase (Adenine-Specific) chemistry
Subjects
Details
- Language :
- English
- ISSN :
- 1940-087X
- Issue :
- 107
- Database :
- MEDLINE
- Journal :
- Journal of visualized experiments : JoVE
- Publication Type :
- Academic Journal
- Accession number :
- 26862720
- Full Text :
- https://doi.org/10.3791/53620