PRO-cap

PRO-cap

PRO-cap maps RNA polymerase II initiation sites –during RNA transcription with base-pair resolution. This approach is a variation of the Pro-Seq method, which maps Pol II pause sites. A nuclear run-on reaction with biotin-NTP and sarkosyl is carried out on nucleic lysates. Incorporation of the first biotin-NTP halts further elongation of nascent RNA strands by RNA pol II. The RNA strands are extracted and purified through streptavidin pull-down. 3' adaptors are directly ligated to the purified sample prior to another streptavidin purification step. 5' ends are repaired using Antarctic phosphatase and tobacco acid pyrophosphatase before ligating 5' adaptors. Adaptor-flanked RNA fragments are enriched through another streptavidin pull-down process before reverse transcription and PCR amplification. Resultant cDNA strands are then sequenced from the 5' end– and RNA pol II pause sites mapped.

Pros:

• Maps RNA pol II initiation sites with base-pair resolution
• Multiple biotin enrichment steps pre-PCR
• Pause sites mapped using PRO-Seq

Cons:

• Restricted to in-vitro reactions

• PRO-cap: 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. Wang I. X., Core L. J., Kwak H., Brady L., Bruzel A., et al. (2014) RNA-DNA differences are generated in human cells within seconds after RNA exits polymerase II. Cell Rep 6: 906-915