DRB/TTchem-seq method

The DRB/TT_chem-seq was carried out as described in Gregersen et al. ²⁴ in two biological replicates. Briefly, 8 × 10⁶ cells were incubated in 100 μM DRB (Sigma-Aldrich) for 3.5 hours. The cells were then washed twice in PBS and fresh, DRB-free medium was added to restart transcription. The RNA was labelled in vivo with 1 mM 4SU (Glentham Life Sciences) for 10 minutes prior to the addition of TRIzol (Thermo Fisher Scientific), which was used to stop the reaction at the desired time point. Following extraction, 100 μg of RNA was spiked-in with 1 μg 4-thiouracile labelled S. cerevisiae RNA (strain BY4741, MATa, his3D1, leu2D0, met15D0, ura3D0), and then fragmented with NaOH and biotinylated with MTSEA biotin-XXlinker (Biotium). The biotinylated RNA was then purified using μMACS Streptavidine MicroBeads (Miltenyi Biotec) and used for library preparation. The libraries were amplified using the KAPA RNA HyperPrep kit (Roche) with modifications as described in⁹.The fragmentation step was omitted and the RNA, resuspended in FPE Buffer, was denatured at 65°C for 5 min. Two SPRI bead purifications were carried out, with a bead-to-sample volume ratio of 0.95x and 1x, respectively. The libraries were then sequenced with single end 75bp reads on the Hiseq4000, with ~50,000,000 reads per sample.