New Chromatin Run-On Reaction Enables Global Mapping of Active RNA Polymerase Locations in an Enrichment-free Manner

Abstract

The development of a simple and cost-effective method to map the distribution of RNA polymerase II (RNPII) genome-wide at a high resolution is highly beneficial to study cellular transcriptional activity. Here we report a mutation-based and enrichment-free global chromatin run-on sequencing (mGROseq) technique to locate active RNPII sites genome-wide at nearbase resolution. An adenosine triphosphate (ATP) analog named N 6 -allyladenosine triphosphate (a 6 ATP) was designed and could be incorporated into nascent RNAs at RNPII-located positions during a chromatin run-on reaction. By treatment of the run-on RNAs with a mild iodination reaction and subjection of the products to reverse transcription into complementary DNA (cDNA), base mismatch occurs at the original a 6 A incorporation sites, thus making the RNPII locations detected in the high-throughput cDNA sequencing. The mGRO-seq yields both the map of RNPII sites and the chromatin RNA abundance and holds great promise for the study of single-cell transcriptional activity.

N 4-Allylcytidine: a new nucleoside analogue for RNA labelling and chemical sequencing

“…It will be ideal to invent new NTP analogues with a chemical sequencing power for ChRO-seq application because complicated and expensive enrichment step is free and false positive signals brought by immunoprecipitation is largely avoided. 44 The above results reveal that: (i) a 4 CTP is an NTP analogue with chemical sequencing capacity; (ii) a 4 CTP is able to be recognized and utilized by cellular RNA polymerase machineries. Therefore, we proposed that a 4 C-based ChRO-seq (a 4 C-ChRO-seq) would fulfil the hope of enrichment-free characterization of genome-wide RNA polymerase transcription activities.…”

“…The nuclei were isolated as previously described. 44 Chromatin run-on reaction and RNA extraction Briefly, 100 mL HeLa nuclei (B5 Â 10 6 ) were mixed with 10 mL 5 M NaCl by pipetting up and down until the solution became clear. Afterwards, an equal volume of nuclease-free water was added, and chromatin was pelleted by centrifugation at 12 000 rpm for 30 s. The chromatin pellet was washed three times with 500 mL 50 mM Tris-HCl (pH = 7.5), followed by resuspension in 100 mL glycerol storage buffer.…”