Nascent RNA profiles of WT and MED23-KD 293T cells

To profile the nascent RNAs, we performed 4SU-seq. Briefly, WT or MED23-KD 293T cells were seeded at 50% confluence in a 10 cm dish and cultured overnight. The newly transcribed RNAs in living cells were then transiently labelled with 4SU at a final concentration of 1 mM for 15 minutes. The labelled cells were harvested immediately with TRIzol. Total RNA was extracted with phenol/chloroform/isoamyl alcohol. The 4SU-labelled RNAs were biotinylated and enriched using streptavidin beads. Finally, the enriched nascent RNAs were subjected to library construction using a NEBNext Ultra Directional RNA Library Prep Kit for Illumina for sequencing. MED23 was knocked down by CRISPR-Cas9 in 293T cells. WT, MED23KD-1 and MED23KD-2 293T cells with two replicates were subjected to 4SU-seq.