"PRO-seq uses biotin-labeled ribonucleotide triphosphate analogs (biotin-NTP) for nuclear run-on reactions, allowing the efficient affinity purification of nascent RNAs for high throughput sequencing from their 3’ ends (Figs. 1A, S1A). Supplying only one of the four biotin-A/C/G/UTP restricts Pol II to incorporate a single or at most a few identical bases, resulting in sequence reads that have the same 3’ end base within each library (table S1). Moreover, the incorporation of the first biotin-base inhibits further transcript elongation, ensuring base-pair resolution (fig. S2)."
Excerpts from "Genome-Wide Control of RNA Polymerase II Activity by Cohesin"
"PRO-seq varies from GRO-seq in that biotin-labeled ribonucleotides are used to allow run-on for a nucleotide or two, instead of the longer run-on with BrUTP used in GRO-seq. PRO-seq, like GRO-seq [17], is highly sensitive, and unlike ChIP, does not depend on crosslinking efficiency or antibody specificity, and detects elongation-competent Pol II regardless of the phosphorylation status. Nuclei were isolated under conditions of ribonucleotide depletion to halt transcription, but leave Pol II transcriptionally engaged. The nascent RNA transcripts produced upon restart of transcription were used to generate a cDNA library for high-throughput sequencing. Inclusion of sarkosyl in the run-on transcription reaction prevents new transcription initiation, so that only Pol II that is already transcriptionally engaged is detected, and gene body and promoter paused Pol II are detected with equal efficiency [17]"
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