title: The Application of DamID to Identify Peripheral Gene Sequences in Differentiated and Primary Cells.
abstract: The nuclear envelope interacts extensively with chromatin, though with differences in degree and specificity in different cell types. However, identifying the specific genome sequences associated with individual nuclear envelope associated proteins, particularly nuclear membrane proteins and lamins, has been particularly difficult due to their inherent insolubility and interconnectivity. DamID is a powerful tool developed to bypass many of the inherent difficulties with identifying nuclear envelope protein-chromatin interactions and, as more tissue culture cell types derived from different tissues are examined by DamID, it is increasingly apparent that there are distinct patterns of genome organization in differentiated cell types. However, in applying DamID to both more diverse and/or differentiated cell types a number of technical caveats to the method have been observed which must be circumvented to ensure high quality data is generated. Here we elaborate a detailed methodology to adapt DamID to novel cell types, in particular differentiated cells in culture. Moreover, we highlight heretofore largely ignored variations in the PCR amplified DNA products generated by the DamID procedure and the consequences they have for downstream analysis steps. Thus, the methods described here should serve as a useful resource to researchers new to DamID as well as readily allow its application to an expanded set of cell types and conditions.
authors.affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Kings Buildings, Michael Swann Building, Room 5.22, Max Born Crescent, Edinburgh, EH9 3BF, UK.
authors.forename: Michael I
authors.initials: MI
authors.lastname: Robson
authors.affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Kings Buildings, Michael Swann Building, Room 5.22, Max Born Crescent, Edinburgh, EH9 3BF, UK. e.schirmer@ed.ac.uk.
authors.forename: Eric C
authors.initials: EC
authors.lastname: Schirmer
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000235:genetics*; D002460:Cell Line; D002843:Chromatin / Q000235:genetics / Q000378:metabolism; D002874:Chromosome Mapping / Q000379:methods; D019295:Computational Biology / Q000379:methods; D019175:DNA Methylation; D005822:Genetic Vectors / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D008780:Methyltransferases / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D024510:Muscle Development / Q000235:genetics; D032446:Myoblasts / Q000378:metabolism; D009685:Nuclear Envelope / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D021141:Nucleic Acid Amplification Techniques; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D014161:Transduction, Genetic
chemical_list: D002843:Chromatin; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D008780:Methyltransferases; C022556:mRNA (2'-O-methyladenosine-N6-)-methyltransferase
keywords: Bacterial dam methylase; DamID; Lamin B1; Myoblasts; Myogenesis; Myotubes; Nuclear envelope; Nuclear lamina