PRO-Seq

PRO-Seq

Precision nuclear run-on sequencing (PRO-Seq) maps RNAP active sites with base-pair resolution. This approach is similar to GRO-Seq, but it provides the added benefit of single-base resolution. RNAPII initiation sites can be mapped using a modified protocol named PRO-cap.

In PRO-seq, a run-on reaction is carried out with biotin-NTPs (either all 4, or one with additional unlabeled NTPs to reduce cost) and sarkosyl, is carried out in isolated nuclei or permeabilized cells. Incorporation of the single biotin-NTP halts further elongation of nascent RNA strands by RNAPII. The RNA strands are extracted, fragmented, and purified through streptavidin pull-down. Next, 3' adapters are ligated directly to the purified sample before another streptavidin purification step. The 5' ends are repaired using TAP and PNK before ligating 5' adapters. The adapter-flanked RNA fragments are enriched through another streptavidin pull-down process before RT and PCR amplification. The resultant cDNA strands are sequenced from the 3' end.

Pros:

• Maps RNAPII pausing sites with base-pair resolution
• Multiple biotin enrichment steps before PCR
• Initiation sites can be mapped using a PRO-cap variant protocol

Cons:

• Unable to detect arrested or backtracked RNAPII complexes¹

• PRO-Seq: Kwak H., Fuda N. J., Core L. J. and Lis J. T. (2013) Precise maps of RNA polymerase reveal how promoters direct initiation and pausing. Science 339: 950-953
• 1. Weber C. M., Ramachandran S. and Henikoff S. (2014) Nucleosomes are context-specific, H2A.Z-modulated barriers to RNA polymerase. Mol Cell 53: 819-830

1. Brent M. R. Past Roadblocks and New Opportunities in Transcription Factor Network Mapping. Trends Genet. 2016;32:736-750
2. Engreitz J. M., Haines J. E., Perez E. M., et al. Local regulation of gene expression by lncRNA promoters, transcription and splicing. Nature. 2016;539:452-455

1. Wang I. X., Core L. J., Kwak H., et al. RNA-DNA differences are generated in human cells within seconds after RNA exits polymerase II. Cell Rep. 2014;6:906-915
2. Danko C. G., Hyland S. L., Core L. J., et al. Identification of active transcriptional regulatory elements from GRO-seq data. Nat Methods. 2015;
3. Core L. J., Martins A. L., Danko C. G., Waters C. T., Siepel A. and Lis J. T. Analysis of nascent RNA identifies a unified architecture of initiation regions at mammalian promoters and enhancers. Nat Genet. 2014;
4. Pagano J. M., Kwak H., Waters C. T., et al. Defining NELF-E RNA binding in HIV-1 and promoter-proximal pause regions. PLoS Genet. 2014;10:e1004090