A new method called global run-on sequencing (GRO-seq) was developed to map and quantify the position, amount, and orientation of transcriptionally engaged RNA polymerases genome-wide. This method involves nuclear run-on assays (NRO) to extend nascent RNAs associated with transcriptionally engaged polymerases under conditions where new initiation is prohibited. A ribonucleotide analog, [5-bromouridine 5'-triphosphate (BrUTP)], was added to BrU-tagged nascent RNA during the run-on step, allowing for the isolation and sequencing of NRO-RNA. The resulting data revealed that promoter-proximal polymerase resides on approximately 30% of human genes, transcription extends beyond pre-messenger RNA 3' cleavage, and antisense transcription is prevalent. Most promoters have an engaged polymerase upstream and in an orientation opposite to the annotated gene, which is associated with active genes but does not elongate effectively beyond the promoter. These results suggest that the interplay between polymerases and regulators over broad promoter regions dictates the orientation and efficiency of productive transcription. GRO-seq data showed that the density of reads peaks near the transcription start site (TSS) in both sense and antisense directions. Alignment of GRO-seq reads to annotated 3' ends of genes revealed a broad peak that extends downstream of polyadenylation sites. The data also showed that divergent transcription is prevalent, with 55% of all genes displaying significant divergent transcription within 1 kb upstream of sense-oriented promoter-proximal peaks. These divergent polymerases are associated with active genes but do not productively elongate. The results indicate that divergent transcription is a mark for most active promoters. The study also found that gene activity, pausing, and divergent transcription correlate with each other and with promoters containing CpG islands. The findings suggest that divergent transcription may play a role in transcriptional regulation by facilitating access of transcription factors to control elements upstream of core promoters. The study also highlights the importance of histone modifications in transcriptional regulation and the need for further research to understand the functions of divergent transcription.A new method called global run-on sequencing (GRO-seq) was developed to map and quantify the position, amount, and orientation of transcriptionally engaged RNA polymerases genome-wide. This method involves nuclear run-on assays (NRO) to extend nascent RNAs associated with transcriptionally engaged polymerases under conditions where new initiation is prohibited. A ribonucleotide analog, [5-bromouridine 5'-triphosphate (BrUTP)], was added to BrU-tagged nascent RNA during the run-on step, allowing for the isolation and sequencing of NRO-RNA. The resulting data revealed that promoter-proximal polymerase resides on approximately 30% of human genes, transcription extends beyond pre-messenger RNA 3' cleavage, and antisense transcription is prevalent. Most promoters have an engaged polymerase upstream and in an orientation opposite to the annotated gene, which is associated with active genes but does not elongate effectively beyond the promoter. These results suggest that the interplay between polymerases and regulators over broad promoter regions dictates the orientation and efficiency of productive transcription. GRO-seq data showed that the density of reads peaks near the transcription start site (TSS) in both sense and antisense directions. Alignment of GRO-seq reads to annotated 3' ends of genes revealed a broad peak that extends downstream of polyadenylation sites. The data also showed that divergent transcription is prevalent, with 55% of all genes displaying significant divergent transcription within 1 kb upstream of sense-oriented promoter-proximal peaks. These divergent polymerases are associated with active genes but do not productively elongate. The results indicate that divergent transcription is a mark for most active promoters. The study also found that gene activity, pausing, and divergent transcription correlate with each other and with promoters containing CpG islands. The findings suggest that divergent transcription may play a role in transcriptional regulation by facilitating access of transcription factors to control elements upstream of core promoters. The study also highlights the importance of histone modifications in transcriptional regulation and the need for further research to understand the functions of divergent transcription.