NET-Seq

mNET-Seq

mNET-Seq generates profiles of nascent RNA and cotranscriptional RNA processing associated with different C-terminal domain (CTD) phosphorylation states throughout the whole genome. mNET-Seq is able to provide precise sequence reads of RNAPII active sites during transcript elongation and also RNA processing intermediates. First, elongating RNAPII complexes are isolated through chromatin fractionation. They are digested with MNase, breaking down all exposed DNA while leaving RNA strands protected by RNAPII or spliceosomes intact. The RNAPII complexes are immunoprecipitated using RNAPII antibodies and 5' phosphorylated by T4 PNK. Next, 3' linkers are ligated to the 3' hydroxyl end of the RNA strand still embedded within RNAPII. They are also incubated with radioactive ATP to facilitate size selection. Nascent RNA strands are size-selected for 35–100 nt, processed into cDNA sequencing libraries, and sequenced. The use of various RNAPII antibodies during purification raises the versatility and specificity of the technique in targeting CTDs of RNAPII.

Pros:

• Maps nascent RNA strands and cotranscriptional RNA processing during RNAPII elongation with phosphorylated CTDs
• Able to detect sense and antisense transcripts at TSS¹
• No crosslinking—eliminates introduction of artifactual interactions
• MNase digestion is specific and efficient
• Various RNAPII-specific antibodies can be used to increase targeting accuracy

Cons:

• Nascent RNAs shorter than 35 nt cannot be detected reliably
• RNA can degrade during RNAPII immunoprecipitation
• mNET-Seq peaks might be obscured by peaks from cotranscriptional RNA cleavage
• PCR amplification may give rise to peaks from amplification bias.

• mNET-Seq: Nojima T., Gomes T., Grosso A. R., et al. Mammalian NET-Seq Reveals Genome-wide Nascent Transcription Coupled to RNA Processing. Cell. 2015;161:526-540
• 1. Nojima T., Gomes T., Carmo-Fonseca M. and Proudfoot N. J. Mammalian NET-seq analysis defines nascent RNA profiles and associated RNA processing genome-wide. Nat Protoc. 2016;11:413-428

1. Mellor J., Woloszczuk R. and Howe F. S. The Interleaved Genome. Trends Genet. 2016;32:57-71
2. Zaborowska J., Egloff S. and Murphy S. The pol II CTD: new twists in the tail. Nat Struct Mol Biol. 2016;23:771-777

1. Nojima T., Gomes T., Grosso A. R., et al. Mammalian NET-Seq Reveals Genome-wide Nascent Transcription Coupled to RNA Processing. Cell. 2015;161:526-540