Edinburgh Research Explorer

The Application of DamID to Identify Peripheral Gene Sequences in Differentiated and Primary Cells

Research output: Chapter in Book/Report/Conference proceedingChapter (peer-reviewed)

Related Edinburgh Organisations

Original languageEnglish
Title of host publicationThe Nuclear Envelope
Subtitle of host publicationMethods and Protocols
EditorsSue Shackleton, Philippe Collas, Eric C. Schirmer
Place of PublicationNew York
PublisherHumana Press
ChapterPart IV
Pages359-86
Number of pages28
Volume1411
Edition1st
ISBN (Electronic)9781493935307
ISBN (Print)9781493935284
DOIs
StatePublished - 5 May 2016

Publication series

NameMethods in Molecular Biology
PublisherHumana Press
ISSN (Print)1064-3745

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.

    Research areas

  • Animals, Cell Differentiation, Cell Line, Chromatin, Chromosome Mapping, Computational Biology, DNA Methylation, Genetic Vectors, High-Throughput Nucleotide Sequencing, Humans, Lentivirus, Methyltransferases, Mice, Muscle Development, Myoblasts, Nuclear Envelope, Nuclear Proteins, Nucleic Acid Amplification Techniques, Recombinant Fusion Proteins, Transduction, Genetic, Bacterial dam methylase, DamID, Lamin B1, Myogenesis, Myotubes, Nuclear lamina

ID: 76511407