PRO-Seq
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.
Advantages:
- 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
Disadvantages:
- Unable to detect arrested or backtracked RNAPII complexes (Weber et al., 2014)
- Limited to in vitro reactions
Reagents:
Illumina Library prep and Array Kit Selector
Reviews:
Brent M. R. Past Roadblocks and New Opportunities in Transcription Factor Network Mapping. Trends Genet. 2016;32:736-750
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
References:
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
Danko C. G., Hyland S. L., Core L. J., et al. Identification of active transcriptional regulatory elements from GRO-seq data. Nat Methods. 2015;
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;
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
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History: PRO-Seq
Revision by sbrumpton on 2017-06-21 07:50:24 - Show/Hide
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.
Advantages:- 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
Disadvantages:- Unable to detect arrested or backtracked RNAPII complexes (Weber et al., 2014)
- Limited to in vitro reactions
Reagents:Illumina Library prep and Array Kit SelectorReviews:Brent M. R. Past Roadblocks and New Opportunities in Transcription Factor Network Mapping. Trends Genet. 2016;32:736-750Engreitz J. M., Haines J. E., Perez E. M., et al. Local regulation of gene expression by lncRNA promoters, transcription and splicing. Nature. 2016;539:452-455References: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;6:906-915Danko C. G., Hyland S. L., Core L. J., et al. Identification of active transcriptional regulatory elements from GRO-seq data. Nat Methods. 2015;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;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