4 thio uracil 4su

Two biological replicates of K562 cells were diluted 24 h before the experiment was performed. Per replicate, 3.6 × 10⁷ cells in growth medium were labeled at a final concentration of 500 µM 4-thio-uracil (4sU; Sigma-Aldrich) and incubated in 5% CO₂ for 5 min at 37°C. Exactly after 5 min of labeling, cells were harvested at 1500g for 2 min at 37°C. Total RNA was extracted from K562 cells using QIAzol according to the manufacturer's instructions except for the addition of 150 ng RNA spike-in mix (Schwalb et al. 2016 (link)) together with QIAzol. To isolate poly(A) RNA from 75 µg of total RNA, two subsequent rounds of purification by Dynabeads Oligo (dT)₂₅ (Invitrogen) were performed. Purification based on the manufacturer's instructions was performed twice, using 1 mg of Dynabeads Oligo (dT)₂₅ beads for the first round and 0.5 mg for the second round of purification. The quality of polyadenylated RNA selection was assessed using RNA ScreenTape on a TapeStation (Agilent). Sequencing libraries were prepared using the NuGEN ovation universal RNA-seq kit according to the manufacturer's instructions. Fragments were amplified by 10 cycles of PCR and sequenced on an Illumina NextSeq 550 in paired-end mode with a 75-bp read length.

TT-seq was performed as described (Schwalb et al. 2016 (link)) with an ERCC spike-in (three unlabeled and three 4sU-labeled) for normalization (Topal et al. 2019 (link)). Two biological replicates for each strain were grown to 1.5 × 10⁷ cells and labeled with 2.5 mM 4-thiouracil (4su) (Sigma-Aldrich) for 10 min. Cells were harvested by centrifugation at 3000g for 2 min and RNA was extracted by hot acid phenol. RNA was fragmented to <1.5 kb and 4sU-labeled RNA was purified by streptavidin beads. Library prepareation and sequencing were completed as described for RNA-seq. TT-seq data were processed and analyzed as described in the Supplemental Material.
Western blot and flow cytometry are described in the Supplemental Material.