Transcriptome profiling of 4-thiouracil labeling

A fresh plate (YES) was inoculated from glycerol stock. An overnight culture was inoculated (YES medium: 0.5% w/v yeast extract (Difco), 225 mg/l each of adenine, histidine, leucine, uracil, and lysine hydrochloride, 3.0% glucose) from a single colony and grown at 30°C. In the morning, a 120 ml culture (YEA medium: 0.5% w/v yeast extract (Difco), 75 mg/l adenine, 3.0% glucose) was started at OD₆₀₀ 0.1 and grown to OD₆₀₀ of 0.8 at 32°C in a water bath at 150 rpm. 4‐thiouracil was added to 110 ml of culture at 5 mM final concentration. About 20 ml of samples was taken out after 2, 4, 6, 8, and 10 min. Each sample was centrifuged immediately at 32°C, at 3,500 rpm for 1 min. The supernatant was discarded, and the pellet was frozen in liquid nitrogen. All experiments were performed in two independent biological replicates. Total RNA was extracted, and samples were DNase digested with Turbo DNase (Ambion). Labeled RNA was purified as published (Sun et al, 2012).