Mapping microRNA-target interactions in abundant cell types with Spy3-AGO2 Pull-down [CLIP-seq]

Studies have demonstrated that T6B can selectively de-repress miRNA targets in non-neuronal cells. However, since neurons may have a different expression profile of miRNA pathway genes as well as other binding partners of TNRC6, we sought to validate the intended function of in neurons. The first step was to identify bona fide miRNA targets in PNs, we performed AGO2 crosslinking and immunoprecipitation followed by sequencing (CLIPseq). To identify the miRNA-target interactions occuring in cultured cortical pyramidal neurons (PNs),we dissected and dissociated cortices from E18-18.5 embryos. After 14 days in culture, we collected the cells for AGO2 CLIPseq. CLIPseq studies were performed by Eclipse Bioinnovations Inc (San Diego, www.eclipsebio.com) according to the published single-end seCLIP protocol (Van Nostrand et al., 2017).