PRO-seq was performed according to the protocol (38) with minor modifications. In the nuclear run-on reaction using MB with spike-in Drosophila S2, all four biotinylated nucleotides were used at 25 μM each final concentration. RppH (RNA 5’ pyrophosphohydrolase) (NEB) was used to remove the 5′ RNA cap. DNA Libraries were size-selected by AMPure XP beads (Beckman Coulter) and sequenced on a NextSeq 500. Adaptors were removed from raw reads by cutadapt 1.14 (60). Reads were trimmed from the 3′ end with removing low-quality bases using Trimmomatic 0.33 (61), requiring a read length of 16 to 36 bp. Reads derived from ribosomal RNA were filtered out by mapping reads on human and fly ribosomal DNA. The remaining reads were mapped on human hg19 or fly dm3 genome using Bowtie 1.1.2 (45) with options -m 1 -v 2. The 5′ ends of the reads were taken using bedtools genomecov 2.25 (62) with options -strand -bg -5, and strands of reads were then swapped. Read counts were normalized by spike-in aligned reads. Normalized read counts were divided by total aligned million reads of WT sample. Metagene profiles of PRO-seq were generated by deepTools 3.0.0 computeMatrix and plotProfile (63).

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