Transcriptome Profiling of K562 Cells

A detailed step-by-step protocol has been deposited in the protocols.io repository (Gressel et al. 2019a ). TT-seq and RNA-seq were performed in 2 biological replicates including RNA spike-ins. Briefly, experiments were performed using $5\times {10}^7$ K562 cells per biological replicate. Cells were kept at optimal growth conditions and supplemented with 5 µg mL⁻¹ of α-amanitin or solvent (water) for 8 h. After 7 h 55 min, a 4-thiouridine (4sU) labeling pulse (Sigma-Aldrich, T4509) was applied for 5 min using 500 µM (see Supplementary material S3). Total RNA was isolated with the QIAzol reagent (# 79306) according to manufacturer’s instructions except for the addition of 150 ng of RNA spike-in pool with QIAzol reagent as previously described (Schwalb et al. 2016 (link); Gressel et al. 2019a ). The Ovation Universal RNA-Seq System (NuGEN) was used for strand-specific library preparation as described (Gressel et al. 2019b (link)). Purified cDNA libraries were analyzed by Fragment Analyzer prior to Illumina sequencing. Sequencing was performed on a HiSeq 2500 (Illumina) in paired-end mode with 50-bp read length.