Genome-wide method for mapping of engaged rna polymerases quantitatively and at high resolution

A method for identifying a transcription start site in the genome of a cell of interest comprising the steps of:

• 1) permeabilizing the cell of interest or isolating the nucleus from the cell of interest;
• 2) performing a nuclear run-on (NRO) reaction with the permeabilized cell or the isolated nucleus, wherein a purifiable nucleotide analog is added to the NRO reaction;
• 3) 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;
• 4) isolating NRO-RNA from the NRO reaction;
• 5) hydrolyzing the NRO-RNA isolated from the NRO reaction to optimize resolution of polymerase location;
• 6) selecting hydrolyzed NRO-RNA with a solid support to obtain a highly enriched and purified fraction of the hydrolyzed NRO-RNA;
• 7) enzymatically repairing the hydrolyzed NRO-RNA;
• 8) selecting capped NRO-RNAs through enzymatic enrichment by the oligo-capping method;
• and
• 9) ligating the hydrolyzed NRO-RNA to compatible adapter oligos.

A method for identifying the position of an active site of an engaged RNA polymerase in the genome of a cell of interest comprising the steps of:

• 1) permeabilizing the cell of interest or isolating the nucleus from the cell of interest;
• 2) hydrolyzing RNA in the permeabilized cell or the isolated nucleus with an RNase;
• 3) performing a nuclear run-on (NRO) reaction with the permeabilized cell or the isolated nucleus, wherein a purifiable nucleotide analog is added to the NRO reaction;
• 4) 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;
• 5) isolating NRO-RNA from the NRO reaction;
• 6) selecting hydrolyzed NRO-RNA with a solid support to obtain a highly enriched and purified fraction of the hydrolyzed NRO-RNA;
• 7) enzymatically repairing the hydrolyzed NRO-RNA by removing a 5′ cap from the NRO-RNA and adding a 5′-P to the NRO-RNA;
• and
• 8) ligating the hydrolyzed NRO-RNA to compatible adapter oligos.

A method for mapping a site of co-transcriptional cleavage that delineates the 3′ end of an mRNA comprising the steps of:

• 1) permeabilizing the cell of interest or isolating the nucleus from the cell of interest;
• 2) 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;
• 3) 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;
• 4) isolating NRO-RNA from the NRO reaction;
• 5) optionally hydrolyzing the NRO-RNA isolated from the NRO reaction to optimize resolution of polymerase location;
• 6) selecting hydrolyzed NRO-RNA with a solid support to obtain a highly enriched and purified fraction of the hydrolyzed NRO-RNA;
• 7) enzymatically repairing the hydrolyzed NRO-RNA removing a 3′-P from the hydrolyzed NRO-RNA;
• and
• 8) ligating the hydrolyzed NRO-RNA to compatible adapter oligos.