Mapping microRNA-target interactions in rare cell types with Spy3-AGO2 Pull-down
To illuminate the molecular mechanisms driving neuronal differentiation we generated a mouse line amenable to mapping miRNA-target interactions in rare cell types. Biochemical approaches to purify AGO2-miRNA-target complexes have successfully mapped MTIs in abundant populations of neurons. However, due to their technical complexity and high background, these approaches are not suitable for mapping interactions in rare cell populations such the many neuronal subtypes that compose the mammalian brain. We therefore generated a mouse line with a conditional SpyTag3, which is small and offers near-infinite affinity for pull-downs, in the endogenous Ago2 gene. We then developed a method Spy3-AGO2 pull-down and sequencing (SAPseq), which we have used to accurately map miRNA-target interactions in developing Purkinje cells, a rare population of cells in the cerebellum. Overall design: To identify the miRNA-target interactions occuring in developing cerebellar Purkinje cells (PCs) we delivered PC-specific AAV expressing Cre to conditional Spy3-AGO2 mouse pups at P0 and then isolated their cerebella at P7 and P15. We then performed AGO2 pull-down using a SpyCatcher3 resin followed by RNA sequencing to generate miRNA libraries and target libraries