NET-Seq
NET-Seq
NET-Seq detects nascent, actively transcribed RNAPII RNAs, through the capture of 3' RNA. In this method, the RNAPII elongation complex is immunoprecipitated, and RNA is extracted and reverse-transcribed to cDNA. Deep sequencing of the cDNA allows for 3'-end sequencing of nascent RNA, providing nucleotide-resolution mapping of transcripts.
Pros:
- Mapping of nascent RNA-bound protein
- Transcription is mapped at nucleotide resolution
Cons:
- Antibodies not specific to the target will precipitate nonspecific complexes
- Requires nascent RNAs of at least 18 nt1
- Hrdlickova R., Toloue M. and Tian B. RNA-Seq methods for transcriptome analysis. Wiley Interdiscip Rev RNA. 2017;8:n/a-n/a
- Murakawa Y., Yoshihara M., Kawaji H., Nishikawa M., Zayed H., et al. Enhanced Identification of Transcriptional Enhancers Provides Mechanistic Insights into Diseases. Trends Genet. 2016;32:76-88
- Zhang J. and Landick R. A Two-Way Street: Regulatory Interplay between RNA Polymerase and Nascent RNA Structure. Trends Biochem Sci. 2016;41:293-310
- Liu X., Kraus W. L. and Bai X. Ready, pause, go: regulation of RNA polymerase II pausing and release by cellular signaling pathways. Trends Biochem Sci. 2015;40:516-525
- Lemay J. F. and Bachand F. Fail-safe transcription termination: Because one is never enough. RNA Biol. 2015;12:927-932
- Harlen K. M., Trotta K. L., Smith E. E., Mosaheb M. M., Fuchs S. M. and Churchman L. S. Comprehensive RNA Polymerase II Interactomes Reveal Distinct and Varied Roles for Each Phospho-CTD Residue. Cell Rep. 2016;15:2147-2158
- Mayer A. and Churchman L. S. Genome-wide profiling of RNA polymerase transcription at nucleotide resolution in human cells with native elongating transcript sequencing. Nat Protoc. 2016;11:813-833
- Imashimizu M., Takahashi H., Oshima T., et al. Visualizing translocation dynamics and nascent transcript errors in paused RNA polymerases in vivo. Genome Biol. 2015;16:98