APEX2 RNA Proximity Labeling in Mammalian Cell Lines With Low Biotin Permeability

The subcellular localization of RNA plays a critical role in various biological processes, including development and stress response. Proximity labeling eases the detection of localized transcripts and protein enrichment compared to previous techniques that rely on biochemical isolation of subcellular structures. The rapid reaction and small labeling radius of APEX2 make it an attractive alternative to other proximity labeling approaches, such as BioID. However, we found that standard protocols for APEX proximity labeling fail in human induced pluripotent stem cells. Moreover, standard protocols yield heterogeneous labeling of biomolecules across single cells in MCF10A breast epithelial cells. Our results indicate that low biotin permeability in these cell lines is the main cause for failed or inefficient labeling. This protocol outlines improved labeling by combining the rapid hydrogen peroxide-driven APEX2 reaction with the addition of a mild detergent during biotin incubation. This adaptation leads to efficient proximity labeling in hiPSCs and more homogeneous biotinylation across single cells in MCF10As. The adapted protocol extends the use of APEX2 proximity labeling to cell lines with poor biotin permeability. Key features • Builds on methods developed by the Ting Lab [1] and the Ingolia Lab [2] for proximity labeling of transcripts using the enzyme APEX2. • Focuses on cell lines with low biotin permeability, like human induced pluripotent stem cells, by including a mild detergent to increase biotin uptake. • Includes controls for nonspecific molecular localization and statistical methods for processing of resulting sequencing data.