Total RNA was extracted from cells with Trizol (Thermo Fisher Scientific). Drosophila RNA from 4sU labeled S2 cells were added to samples (1:50) for spike-in. RNA samples were fragmented by adding 1 M NaOH (0.2 M final concentration) and incubating on ice for 20 min. Reaction was stopped by adding 1 M Tris buffer (pH 6.8) (0.5 M final concentration). RNA samples were biotinylated with MTSEA biotin-XX (Biotium) and incubated in the dark for 30 min at room temperature. RNA was purified with RNA clean-up kit (500 μg) (New England Biolabs). Biotinylated RNA was enriched with hydrophilic streptavidin magnetic beads (New England Biolabs). Ribosome RNA (rRNA) was removed with NEB rRNA removal module (New England Biolabs). Libraries were prepared with NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs).