3’NT Method
3′ End of Nascent Transcripts
The 3’NT method sequences nascent RNA transcripts to map the positions of elongating and arrested RNAPII complexes at nucleotide resolution (Weber et al., 2014). This method effectively isolates the RNAPII-chromatin complex by capitalizing on its ability to remain bound to the DNA strand in the presence of high salt, urea, detergents, and polyanions.Transcription is arrested in the sample while both the cells and nuclei are lysed. The nascent RNA strands are isolated from the purified RNAPII-chromatin complex and enriched for mRNA by selecting for strands with 5′ 7-methylguanosine caps. cDNA libraries are prepared from nascent mRNA strands by following the protocol for native elongating transcript sequencing (NET-Seq), with slight modifications. The resulting cDNA libraries are sequenced to determine the precise locations of RNAPII during each experimental condition.
Advantages:
- Tracks position of elongating and arrested RNAPII throughout the whole genome
- Does not require transgenes, solubilization, or immunopurification
Disadvantages:
- Unable to interrogate the relationship between RNAPII C-terminal domain modifications and nascent RNA strands (Nojima et al., 2015)
Reagents:
Illumina Library prep and Array Kit Selector
Reviews:
Zentner G. E. and Henikoff S. High-resolution digital profiling of the epigenome. Nat Rev Genet. 2014;
Teves S. S., Weber C. M. and Henikoff S. Transcribing through the nucleosome. Trends Biochem Sci. 2014;39:577-586
References:
Weber C. M., Ramachandran S. and Henikoff S. Nucleosomes are context-specific, H2A.Z-modulated barriers to RNA polymerase. Mol Cell. 2014;53:819-830
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History: 3’NT Method
Revision by sbrumpton on 2017-06-21 07:50:23 - Show/Hide
3' End of Nascent Transcripts
The 3'NT method sequences nascent RNA transcripts to map the positions of elongating and arrested RNAPII complexes at nucleotide resolution (Weber et al., 2014). This method effectively isolates the RNAPII-chromatin complex by capitalizing on its ability to remain bound to the DNA strand in the presence of high salt, urea, detergents, and polyanions.Transcription is arrested in the sample while both the cells and nuclei are lysed. The nascent RNA strands are isolated from the purified RNAPII-chromatin complex and enriched for mRNA by selecting for strands with 5' 7-methylguanosine caps. cDNA libraries are prepared from nascent mRNA strands by following the protocol for native elongating transcript sequencing (NET-Seq), with slight modifications. The resulting cDNA libraries are sequenced to determine the precise locations of RNAPII during each experimental condition.
Advantages:- Tracks position of elongating and arrested RNAPII throughout the whole genome
- Does not require transgenes, solubilization, or immunopurification
Disadvantages:- Unable to interrogate the relationship between RNAPII C-terminal domain modifications and nascent RNA strands (Nojima et al., 2015)
Reagents:Illumina Library prep and Array Kit SelectorReviews:Zentner G. E. and Henikoff S. High-resolution digital profiling of the epigenome. Nat Rev Genet. 2014;Teves S. S., Weber C. M. and Henikoff S. Transcribing through the nucleosome. Trends Biochem Sci. 2014;39:577-586References:Weber C. M., Ramachandran S. and Henikoff S. Nucleosomes are context-specific, H2A.Z-modulated barriers to RNA polymerase. Mol Cell. 2014;53:819-830