Hippocampi from E16 embryos were dissected individually into Eppendorf tubes and flash frozen. The adult hippocampus from a male mouse was dissected and flash frozen. Adult cortex from a male mouse was flash frozen. Samples were stored at −80°C until lysate preparation.
To prepare lysate, frozen tissue was transferred to a 2 mL Dounce homogenizer filled with ribosome-profiling lysis buffer (10 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 100 mM KCl, 1% Triton-X 100, 2 mM DTT, 100 µg/mL cycloheximide, 500 U/mL RNasein Plus [Promega], and cOmplete Mini EDTA-free Protease Inhibitor Tablets [Roche, 1 tablet/10 mL]) (Subtelny et al. 2014 (link)). Samples were then homogenized with 10 strokes of pestle A followed by 10 strokes of pestle B, taking care not to introduce bubbles into the buffer. Following homogenization, the sample was transferred to two Eppendorf tubes, centrifuged at 1300g for 10 min, and the supernatant was aliquoted and flash frozen at −80°C for use in ribosome profiling, RNA sequencing, and tail sequencing. For RNA and tail sequencing, RNA was extracted by adding five volumes of TRIzol (Thermo Fisher) to the frozen lysate, allowing it to thaw to room temperature, and continuing the preparation according to the manufacturer's instructions.
To prepare lysate, frozen tissue was transferred to a 2 mL Dounce homogenizer filled with ribosome-profiling lysis buffer (10 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 100 mM KCl, 1% Triton-X 100, 2 mM DTT, 100 µg/mL cycloheximide, 500 U/mL RNasein Plus [Promega], and cOmplete Mini EDTA-free Protease Inhibitor Tablets [Roche, 1 tablet/10 mL]) (Subtelny et al. 2014 (link)). Samples were then homogenized with 10 strokes of pestle A followed by 10 strokes of pestle B, taking care not to introduce bubbles into the buffer. Following homogenization, the sample was transferred to two Eppendorf tubes, centrifuged at 1300g for 10 min, and the supernatant was aliquoted and flash frozen at −80°C for use in ribosome profiling, RNA sequencing, and tail sequencing. For RNA and tail sequencing, RNA was extracted by adding five volumes of TRIzol (Thermo Fisher) to the frozen lysate, allowing it to thaw to room temperature, and continuing the preparation according to the manufacturer's instructions.
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