ChIRP and RNA interactome experiments were performed as previously described (24, 26). Antisense oligo probes with 3′ biotin-triethyleneglycol modification (TEG) were designed using the online probe designer on the ChIRP Probe Designer (version 4.2, LGC Biosearch Technologies, Middlesex, UK) and synthesized by Sigma-Aldrich (data S1J). Note that the designed primers were blasted against whole rat genome and do not specifically bind other targets than neuroLNC. The 10 designed probes for neuroLNC were divided into two tiling pools (arbitrarily defined odd and even pools). We also designed 10 LacZ probes, which served as negative controls. We performed the neuroLNC enrichment from the brain of 2-day-old (P2) rats where neuroLNC expression is high. For ChIRP, cerebral cortices were homogenized with a micropestle in 500 μl of ice-cold low-sucrose buffer [0.32 M sucrose, 5 mM CaCl2, 5 mM Mg(Ac)2, 0.1 mM EDTA, 50 mM Hepes (pH 8), 1 mM dithiothreitol (DTT), and 0.1% Triton X-100]. Subsequently, the cortex homogenate was cross-linked with either 3% formaldehyde or 1% glutaraldehyde and sonicated for either 30 or 120 min for either ChIRP MS or ChIRP DNA/RNA, respectively. Enrichment of neuroLNC was tested by qRT-PCR for ChIRP RNA samples. ChIRP-derived RNA and DNA were sequenced, and eluted proteins were analyzed by MS proteomics as previously described (43).

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