Identifying Genomic DNA Sequences Near the Nuclear Lamina Using Proximity Biotinylation with Ascorbate Peroxidase.

Ascorbate peroxidase (APEX2) is an engineered plant-based enzyme that is used for the proximal ligation of biotin onto nearby proteins and nucleic acids. The APEX2 enzyme can be targeted to a specific subcellular compartment (e.g., the nuclear lamina) by creating a fusion between the APEX2 enzyme and a targeting protein (e.g., Lamin B1). We have found that the APEX2 proximity ligation reaction is very robust and can be performed in both living and formaldehyde-fixed cells. The biotinylated proteins and nucleic acids can then be precipitated with streptavidin beads for the identification of proximal proteins, RNA, and DNA. Here, we detail a protocol for the use of an APEX-Lamin B1 fusion protein to identify DNA in the proximity of the nuclear lamina, including lamina-associated domains (LADs).