PRO-cap
PRO-cap
PRO-cap maps RNAPII initiation sites during RNA transcription with base-pair resolution. This approach is a variation of the PRO-Seq method, which maps RNAPII pause sites. A nuclear run-on reaction with biotin-NTP and sarkosyl is carried out on nuclear lysates. Incorporation of the first biotin-NTP halts further elongation of nascent RNA strands by RNAPII. The RNA strands are extracted 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 Antarctic phosphatase and TAP 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 5' end, and RNAPII pause sites are mapped.
Pros:
- Maps RNAPII initiation sites with base-pair resolution
- Multiple biotin enrichment steps before PCR
- Pause sites mapped using PRO-seq
Cons:
- Limited to in vitro reactions
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