Nascent RNA-seq

Cells (10⁸) were treated with 500 μmol/L 4-thiouridine (4-SU) for 1 hour. Cells were lysed using trizol, and RNA was precipitated with ethanol. 4-SU-Incorporated RNA was biotinylated by labeling with 1 mg/mL Biotin-HPDP for 90 min at room temperature. Following chloroform extraction, labeled RNA was separated using magnetic streptavidin beads. Beads were washed using a magnetic μMACS stand before RNA was eluted in two rounds of elution with 100 μL 100 mmol/L dithiothreitol. RNA was purified using a Qiagen RNeasy MinElute kit. Samples were sequenced on a NextSeq 500 using a high 75 v2 sequencing kit. Nascent RNA-seq experiments were carried out in triplicate.