Yeast RNA was prepared by extraction with hot acidic phenol (19 ). Total RNA was extracted, and 4tU-containing mRNA was biotinylated and isolated as previously described (20 (link),21 (link)). To obtain high quality RNA, samples were further subjected to the clean-up protocol with the RNeasy Mini Kit (Qiagene, Cat. #74104), and quality was checked with gel electrophoresis. Nascent RNA was then biotinylated as described (20 (link),21 (link)), using 400 μl (∼40 μg) total RNA and 4 μg MTSEA biotin-XX (Biotium) and purified by MyOne Streptavidin C1 Dynabeads (Invitrogen). The isolated RNA was further purified and concentrated using Qiagen miRNeasy columns (#217084), according to the manufacturer's protocol. The isolated RNA was then prepared for sequencing using the Ovation SoLo or Ovation Universal RNA-seq System kits (Tecan), according to the manufacturer's instructions. Libraries were sequenced on the Illumina NextSeq high output platform using single reads 75 in Institute of Molecular Biology of Academia Sinica