EXPERIMENTAL FIGURE 9-17 Analysis of elongating RNA polymerase II molecules in human fibroblasts. Nuclei from cultured fibroblasts were isolated and incubated in a buffer with a non-ionic detergent that prevents RNA polymerase II from initiating transcription. Treated nuclei were then incubated with ATP, CTP, GTP, and Br-UTP for 5 minutes at 30 °C, a time sufficient to incorporate about 100 nucleotides. RNA was then isolated and broken into fragments of about 100 nucleotides each by controlled incubation at high pH. Specific RNA oligonucleotides were ligated to the 5′ and 3′ ends of the RNA fragments, which were then subjected to reverse transcription. The resulting DNA was amplified by the polymerase chain reaction and subjected to massively parallel DNA sequencing. The sequences determined were aligned to the transcription start sites (TSS) of all known human genes, and the number of sequence reads per kilobase of total sequenced DNA was plotted for 10-bp intervals of sense transcripts (blue) and antisense transcripts (purple). See text for discussion.

[Data from L. J. Core, J. J. Waterfall, and J. T. Lis, 2008, Science 322:1845.]