Precision Nuclear Run-on Sequencing

Precision nuclear run-on sequencing (PRO-Seq) maps RNAPII pause sites with base-pair resolution during RNA transcription (Kwak et al., 2013) (Mahat et al., 2016). 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, 4 separate run-on reactions, each with only 1 type of biotin-NTP and sarkosyl, are carried out on nuclear lysates. 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.


  • Maps RNAPII pausing sites with base-pair resolution
  • Separate run-on reactions limit the addition of nucleotides other than the provided biotin-NTP
  • Multiple biotin enrichment steps before PCR
  • Initiation sites can be mapped using PRO-cap


  • Unable to detect arrested or backtracked RNAPII complexes (Weber et al., 2014)
  • Limited to in vitro reactions


Illumina Library prep and Array Kit Selector


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