, 2004 and Rossner et al., 2006), fluorescence-activated cell sorting (FACS) (Tomomura et al., 2001 and Lobo et al., 2006), or manual sorting (Sugino et al., 2006). These methods are often labor intensive and of low yield. Furthermore, the physical damage and stress inherent to these procedures may alter the physiological state of cells

and likely their gene expression. The recent invention of genetic tagging methods, such as TRAP (Heiman et al., 2008b) and Ribo-tag (Sanz et al., 2009), begin to overcome these obstacles, but these strategies have been limited to the analysis of mRNA expression. Here, we present a novel miRNA tagging and affinity-purification method, miRAP, which can be applied Anti-cancer Compound Library to genetically defined cell types in any complex tissues in mice. This method is based on the fact that mature miRNAs are incorporated into RNA-induced silencing complex (RISC), in which the Argonaute protein AGO2 directly binds miRNAs and their mRNA targets (Hammond et al., 2001). We demonstrate that epitope tagging of AGO2 protein allows direct check details purification of miRNAs from tissue homogenates using antibodies against the engineered molecular tag. We further established

a Cre-loxp binary expression system to deliver epitope-tagged AGO2 (tAGO2) to genetically defined cell types. To demonstrate the feasibility of this approach in the brain, we have analyzed miRNA profiles from five neuron types in mouse cerebral cortex and cerebellum by deep sequencing. Our study reveals the expression of a large fraction of known miRNAs (over 480) which show distinct profiles in second glutamatergic and GABAergic neurons, and subtypes of GABAergic neurons. Our method further detects 23 putative novel miRNAs and also provides evidence for tissue-specific strand selection

of miRNAs and miRNA editing in subset of neuron types. The miRAP method therefore enables a systematic analysis of miRNA expression and regulation in different neuron types in the brain and is generally applicable to other cell types and tissues in mice. Our strategy for molecular tagging and affinity purification is based on the knowledge that mature miRNA is incorporated into the RNA-induced silencing complex (RISC) where the miRNA and its mRNA target interact (Hammond et al., 2001). Argonaute (AGO) proteins are at the core of RISC complex and directly bind miRNAs. AGO immunoprecipitation has been successfully used to isolate miRNAs and their mRNA targets (Easow et al., 2007, Beitzinger et al., 2007, Hendrickson et al., 2008, Zhang et al., 2007, Hammell et al., 2008 and Karginov et al., 2007). Among the four members in the AGO family in human and mouse, only AGO2 exhibits endonuclase activity (Meister et al., 2004 and Ikeda et al., 2006) and is indispensible for Dicer-independent miRNA biogenesis (Cheloufi et al., 2010). We therefore chose to tag AGO2 by fusing GFP and MYC at its N terminus (tAGO2) (Figure 1B).