Trizol and chloroform mixture

Wandering third instars (20 larvae) of each genotype (UH1>FMRP-YFP or Tubulin-GFP) were homogenized in 200 μL of RNase-free lysis buffer (20 mM HEPES, 100 mM NaCl, 2.5 mM EDTA, 0.05% (v/v) Triton X-100, 5% (v/v) glycerol) with 1% β-mercaptoethanol 1× protease inhibitor cocktail (complete mini EDTA-free Tablets, Sigma, 11836170001) and 400U RNase inhibitor (Applied Biosystems, N8080119). The supernatant was collected and diluted to 300 μL to reduce nonspecific binding. Next, the samples were incubated with GFP-trap coupled magnetic agarose beads (Chromotek, GTMA20) for 3 hours at 4°C. The bound beads were washed with lysis buffer (3X, 10 minutes). The bound RNA was purified by incubating the bead-protein-RNA conjugates with a 500-μL TRIzol and chloroform mixture (Ambion, 15596026) for 10 minutes at RT, followed by centrifugation. To precipitate RNA, glycogen (1 μL) and 2-propenol (250 μL) were added to the isolated aqueous layer. Finally, the precipitated RNA was reverse transcribed into single-strand cDNA using the SuperScript VILO cDNA synthesis kit (Thermo Fisher, 11754050) and then subjected to primer-specific PCR, with 2% agarose gels used to analyze the PCR products. All primers used in this study are summarized above in Table 2.