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.
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Abbreviations
- gDNA:
-
Genomic DNA
- NE:
-
Nuclear envelope
- NET :
-
Nuclear envelope transmembrane protein
- PCR :
-
Polymerase chain reaction
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Acknowledgments
The authors would like to thank Jose de las Heras who contributed significantly to the establishment and analysis of DamID in the Schirmer lab, and Bas van Steensel, Carolyn de Graaf, and Job Kind who provided considerable guidance on the method. MIR was supported by a Wellcome Trust Ph.D. Studentship (093854). Funding for this work was provided by Welcome Trust grants 095209 to ECS and 092076 for the Centre for Cell Biology.
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Robson, M.I., Schirmer, E.C. (2016). The Application of DamID to Identify Peripheral Gene Sequences in Differentiated and Primary Cells. In: Shackleton, S., Collas, P., Schirmer, E. (eds) The Nuclear Envelope. Methods in Molecular Biology, vol 1411. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3530-7_23
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DOI: https://doi.org/10.1007/978-1-4939-3530-7_23
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