Protein g agarose bead

NPCs were resuspended in lysis buffer (50 mM Tris pH 7.4, 100 mM NaCl, 1% NP-40, 0.1% SDS and 0.5% sodium deoxycholate) supplemented with 1 × Protease Inhibitor cocktail (Roche) and 80 U of RNAse Inhibitor (Roche). Clarified lysates were pre-cleared with Protein G agarose beads (Life Technologies). Aliquots of the supernatant (equivalent to 5% of supernatant) were saved as input protein and RNA. The remainder of the supernatant was incubated with 10 μg of antibody at 4 °C for 4 h. The protein–RNA–antibody complex was precipitated by incubation with Protein G magnetic beads overnight at 4 °C. Beads were washed twice with lysis buffer and three times with wash buffer (5 mM Tris pH 7.5, 150 mM NaCl, 0.1% Triton X-100). Ten per cent of the bead slurry was reserved for western blot analysis. The remaining bead slurry was resuspended in TRIzol (Life Technologies), and RNA was extracted as per the manufacturer's instructions. Input and immunoprecipitated RNA was converted into cDNA and gene expression was measured with qPCR. RNA immunoprecipitation qPCR studies were performed in biological duplicates. Primer sequences are listed in Supplementary Data 9.

Sciatic nerves or cells were lysed in immunoprecipitation buffer (Cat# 87788, Thermo scientific) with proteinase/phosphatase inhibitor cocktail and incubated with primary antibodies overnight at 4°C with rotation (70 rpm). Protein G agarose beads (Cat# 15920–010, Life technologies) were added for another 2 hour incubation at 4°C. Samples were eluted with Laemmli sample buffer (Cat# 161–0737, Bio-rad), resolved by SDS-PAGE, and analyzed by immunoblot. All antibodies and their titers in this study are listed in S4 Table.

Lysates from cells in culture were prepared by suspending in suspension buffer (0.1 M NaCl, 10 mM Tris-HCl [pH 7.5], 1 mM EDTA [pH 8.0], 1 mM DTT and protease inhibitors) and a same volume of Laemmli buffer (0.1 M Tris-HCl [pH 7.0], 4% SDS, 20% glycerol, 1 mM DTT and protease inhibitors), and boiled for 10 min. Western blotting was performed according to standard procedures and quantified by Image J^®. For statistical analysis, at least three independent immunoblots were analyzed. For co-immunoprecipitation, cells were lysed into cold RIPA 250 buffer (50 mM Tris-HCl [pH 7.4], 250 mM NaCl, 0.5% NonidetP-40, 1 mM EDTA, 1 mM DTT, 10 mM NAM and protease inhibitor cocktail). 2 μg primary antibodies or appropriate control IgGs (Santa Cruz) were added to the lysates and incubated for 2 h on a rocking platform at 4^oC, following incubation with Protein G Agarose beads (Life Technologies) for overnight. The beads were then washed twice with lysis buffer and boiled in SDS sample buffer. Subsequently, the protein suspension was collected and detected by western blotting.