7SK ncRNA as a gene-specific transcriptional repressor in embryonic stem cells (ESCs). (A) qRT-PCR analysis of 7SK and Olig2 total RNA (nascent and processed RNA), and Pou5f1 (Oct4) mRNA 6 hours after nucleofection of ESCs with antisense oligonucleotides (ASOs) targeting the 5′ and 3′ segments of 7SK, with green fluorescent protein (GFP) and scrambled ASOs as control. Error bars represent standard error of the mean (SEM) from 2 to 3 independent experiments. (B) Quantitative reverse transcription (qRT)-PCR analysis of Dll1 and Olig2 total RNA in ESCs 6 hours after nucleofection with 7SK 3′ ASOs. ESCs were grown in serum (Ser-Ser) or 2i/LIF medium (2i-2i), or were switched from 2i/LIF to serum-containing media after nucleofection (2i-Ser). Error bars represent SEM from two independent experiments. (C) RNA sequencing (RNA-seq) read coverage at the Dll1 locus. For this and all other genome browser images, read counts were normalized (see Materials and Methods), averaged over biological replicates, and visualized with Ensembl. The plus (green) and minus (blue) strand reads are displayed in separate tracks. (D) The 50 most significantly upregulated genes after 7SK knockdown (that is, having the lowest P-values) were sorted by fold change. Color scale indicates expression relative to scrambled ASO mean (two biological replicates per ASO, assayed by RNA-seq). (E) Exonic and intronic normalized RNA-seq read counts for Olig2, Irx2, Dll1, c-Myc, Nanog, and Pou5f1 (Oct4), averaged over replicates.