7SK represses upstream divergent RNAs (udRNAs) and long non-coding RNA (lncRNAs) but not their associated transcriptionally active genes, and positive transcription elongation factor b (P-TEFb) is involved in udRNA transcription. (A) Quantitative reverse transcription (qRT)-PCR analysis of Rbm34 and Mettl16 udRNAs 6 hours after nucleofection of embryonic stem cell (ESCs) with scrambled or 7SK 3′ antisense oligonucleotides (ASOs), in the presence or absence of flavopiridol. Error bars represent standard error of the mean (SEM) from two independent experiments. (B) qRT-PCR analysis of udRNAs adjacent to Rbm34, hnRNPL, and Mett1l6, and corresponding mRNAs 6 hours after nucleofection of mouse ESCs with control ASOs or ASOs targeting 7SK. ESCs were grown in serum (Ser-Ser) or 2i/LIF media (2i-2i), or switched from 2i/LIF to serum media after nucleofection (2i-Ser). SEM from two to three independent experiments. (C) qRT-PCR analysis of 7SK total RNA, c-Myc spliced mRNA, and Rbm34 and Mett1l6 udRNAs, 6 hours after nucleofection of ESCs with scrambled of 7SK 3′ ASOs, in the presence or absence of I-BET151. Error bars represent SEM from two to three independent experiments. (D) Box plot depicting log2 fold changes measured by RNA sequencing (RNA-seq) after 7SK knockdown of udRNAs and their associated genes in mouse ESCs. (E) Box plot depicting log2 fold changes measured by RNA-seq after 7SK knockdown in mouse ESCs of 7SK-regulated udRNAs overlapping divergent long intergenic non-coding RNAs (lincRNAs) and their associated genes.