GENOME-WIDE METHOD FOR MAPPING OF ENGAGED RNA POLYMERASES QUANTITATIVELY AND AT HIGH RESOLUTION

摘要

A method is provided for detecting genome-wide transcriptionally-engaged RNA polymerases. The method can also be used to assess status and regulation of gene promoters. The method comprises permeabilizing a cell of interest or isolating the nucleus from a cell of interest; performing a nuclear run-on (NRO) reaction with the permeabilized cell or isolated nucleus, wherein a purifiable nucleotide analog is added to the NRO reaction; optimizing the number of bases traveled by engaged polymerases for high resolution and low bias for nucleotide content of transcribed sequences by limiting a second nucleotide concentration or duration of the NRO reaction; isolating NRO-RNA from the NRO reaction; hydrolyzing the NRO-RNA isolated from the NRO reaction to optimize resolution of polymerase location; selecting hydrolyzed NRO-RNA with a solid support to obtain an enriched, purified fraction of the hydrolyzed NRO-RNA; enzymatically repairing the hydrolyzed NRO-RNA; and ligating the hydrolyzed NRO-RNA to compatible adapter oligos.