7SK ncRNA directly represses a subset of genes with bivalent or active chromatin marks in embryonic stem cells (ESCs), through a mechanism involving positive transcription elongation factor b (P-TEFb). (A) Box plot of RNA sequencing (RNA-seq) gene expression values (reads per kilobase per million (RPKM); see Materials and Methods), averaged over the control antisense oligonucleotide (ASO) samples, for genes that were upregulated (left, red), downregulated (middle, blue) and not significantly altered (right, green) by 7SK knockdown. Data are shown for the set of genes considered for differential expression analysis (see Materials and Methods). (B) Quantitative reverse transcription (qRT)-PCR analysis of 7SK, Olig2, and Hexim1 total RNAs, and for Dll1 and Hes1 nascent RNAs 6 hours after nucleofection of ESCs with scrambled 7SK 3′ ASOs, in the presence or absence of flavopiridol. Error bars represent standard error of the mean (SEM) from two to three independent experiments. (C) Histone modification status in mouse ESCs  for all protein-coding and long intergenic non-coding RNA (lincRNA) genes larger than 1 kb (top), the subset expressed in ESCs (middle; RPKM > 5 in control ASO sample), and the subset directly repressed by 7SK (bottom). Similar results were obtained when data were compared with those of Young et al. (D) Box plots of gene expression values as in panel (A), further stratified by chromatin mark status as in panel (C). *P < 0.05, **P < 0.01; Kolmogorov-Smirnov test.