GSE160073
New Chromatin Run-On Reaction Enables Global Mapping of Active RNA Polymerase Locations in an Enrichment-free Manner
The status of cellular RNA polymerases' locations mirrors global transcriptional activity. A number of methods have been developed to map the distribution of RNA polymerase II (RNPII) genomewide, most of which took advantages of im-munoprecipitations of RNPII complexes or nucleotide analog-labeled nascent RNAs followed by high-throughput sequenc-ing. Given the tedious procedures involved in enrichment and sequencing library construction experiments, development of a simple method that conquers these issues is highly beneficial to the field. Here we report a mutation-based and enrich-ment-free Global chromatin Run-On sequencing (mGRO-seq) technique to map strand-specific RNPII locations ge-nomewide at near base resolution. An ATP analog, named N6-allyladenosine triphosphate (a6ATP), was designed and incor-porated into nascent RNAs at RNPII-located positions during a chromatin run-on assay. The chromatin RNAs were purified and subjected to a mild iodination reaction, which quantitatively transformed a6A into 1,N6-cyclized adenosine (cyc-A). During reverse transcription of RNA to complementary DNA (cDNA), base misincorporation at opposite site of cyc-A oc-curred and mutation reads were generated in the cDNA high-throughput sequencing. Bioinformatics analysis identified the genomewide RNPII location distributions from the characteristic A-mutated sites. mGRO-seq yields both the profile of RNPII sites and the chromatin RNA abundance from one experiment. To sum up, our work demonstrated a new simple and cost-effective method for mapping RNA polymerase locations genomewide, which holds great promise for single-cell char-acterization of RNPII distributions. Overall design: Identification of RNA polymerase active sites in HeLa cell line using mGRO-seq method.