title: Lamin ChIP from Chromatin Prepared by Micrococcal Nuclease Digestion.
abstract: It is now clearly demonstrated that nuclear lamins interact with the genomic DNA and largely contribute to its three-dimensional organization and transcriptional regulation. Emergence of genome-wide mapping techniques such as DamID technology or chromatin immunoprecipitation (ChIP) followed by array hybridization or high-throughput sequencing has allowed the mapping of large lamin-interacting genomic areas called lamina-associated domains. These cover up to 40 % of the genome and are preferentially located in transcriptionally silent heterochromatin at the nuclear periphery. We recently showed that the use of enzymatic rather than physical fragmentation of chromatin in ChIP experiments uncovers new chromatin compartments with features of euchromatin that interacts with A-type lamins. We describe here a detailed ChIP procedure to covalently cross-link protein-DNA, fragment the chromatin fibers by micrococcal nuclease digestion, and solubilize the lamin network with a short sonication pulse prior to immunoprecipitating the lamin-DNA complexes using specific antibodies. Enriched DNA fragments from the lamin-binding sites are then purified as suitable samples for qPCR analysis or high-throughput sequencing.
authors.affiliation: Pathophysiology of Striated Muscles Laboratory, Unit of Functional and Adaptive Biology, University Paris Diderot-UMR CNRS 8251, Paris, France. Isabelle.duband-goulet@univ-paris-diderot.fr.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Duband-Goulet
mesh_terms: D001665:Binding Sites; D002843:Chromatin / Q000235:genetics* / Q000378:metabolism*; D047369:Chromatin Immunoprecipitation / Q000379:methods; D004247:DNA / Q000235:genetics / Q000378:metabolism; D059014:High-Throughput Nucleotide Sequencing; D034882:Lamins / Q000378:metabolism*; D008836:Micrococcal Nuclease / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism; D011485:Protein Binding
chemical_list: D002843:Chromatin; D034882:Lamins; D009687:Nuclear Proteins; D004247:DNA; D008836:Micrococcal Nuclease
keywords: ChIP; Chromatin; Lamin B1; Lamina-associated domain; Lamins A/C; Micrococcal nuclease digestion