design: TT-TimeLapse-seq was performed with 20 million cells per sample labeled with 1 mM 4sU for 5 min. Total RNA was extracted, spiked with 4sU-treated Drosophila RNA, sheared, and biotinylated with MTSEA-biotin-XX. Biotinylated RNA was isolated using streptavidin beads and eluted with DTT. TimeLapse chemistry converted U to C, and libraries were prepared using the Takara Pico Mammalian V3 kit.