7SK knockdown is associated with failed transcriptional termination at specific loci. (A) RNA sequencing (RNA-seq) read coverage plot showing that 7SK knockdown results in increased transcription across an extensive region (box) downstream of Eif4a3, including Cbx4. The plus (green) and minus (blue) strand reads are displayed in separate tracks. (B) Mean change in RNA-seq read coverage around protein-coding genes after 7SK knockdown. Log2 fold changes on the sense (blue) and antisense (red) strands were determined in 500 bp windows, and averaged over genes. (C) Density scatter plots of normalized read counts for protein-coding genes and surrounding regions. Counts from experiments in which ESCs were nucleofected with 5′ and 3′ 7SK ASOs (y-axis) are plotted against counts for ESCs nucleofected with scrambled control ASOs (x-axis), to illustrate the overall change in expression levels after 7SK depletion. Color intensity indicates the density of data points. Read counts were normalized by the total number of mapped reads per sample (see Materials and Methods), incremented by a pseudocount of 1 to enable visualization on a logarithmic scale, and averaged over samples. (D) Heatmap of failed transcriptional termination after nucleofection of ESCs with 7SK 5′ and 3′ ASOs. Each row represents a potential locus of failed transcriptional termination, centered at the 3′ end of the gene (polyadenylation site; PAS) and extending 100 kb upstream and downstream. Genes were ordered by first combining the normalized read distributions about the PAS for the six samples into a single vector for each gene, and are displayed in order from the highest average fold change (at the top) to the lowest.