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2007) using EcoRI and XhoI restriction sites. The dn-tnrc6-gfp fusion gene was then placed downstream of the previously identified her4 promoter (Yeo et al., 2007) or CMV/SP6 (for mRNA synthesis) by performing a gateway LR reaction (Invitrogen) using the p5E-her4, p5E-CMV/SP6 (Tol2kit), pME-eGFP-dn-tnrc6, p3E-polyA (Tol2kit) and pDest-Tol2pA2 (Tol2kit) entry vectors. The full length zebrafish ago2 was amplified from adult brains cDNAs using the Phusion High Fidelity PCR kit (Thermo Scientific). The PCR fragment was cloned into the pCS2+Flag plasmid, The fragment encoding the highly conserved zebrafish tnrc6a GW-I/II was amplified from adult brains cDNAs using the Expand High Fidelity PCR system (Roche). The PCR fragment was cloned into the pME-eGFP no stop vector of the Tol2kit ,
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, Cellular Fractionation of proteins and RNAs The separation and preparation of cytoplasmic, membrane, nuclear soluble, chromatin-bound and cytoskeletal protein extracts was performed with the Subcellular Protein Fractionation Kit for tissues (Thermo Scientific, #87790) according to manufacturer's instructions, starting from 40 adult telencephali (~200mg of tissue) dissected in sterile ice cold PBS. Cytoplasmic and nuclear tissue fractionation and RNA purification was carried out using the Cytoplasmic and Nuclear RNA purification Kit (Norgen Biotek Corp., #37400) according to manufacturer's instructions using either 6 adult telencephali per sample or 4 juvenile (1 month old) whole brains per sample. RT-PCR for gapdh and U2 was performed to verify the purity of the cytoplasmic and nuclear fractions using 18ng total RNA input and using the following reagents: Superscript II kit (Invitrogen) with Random Hexamer Primers
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