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Mammalian NET-seq analysis defines nascent RNA profiles and associated RNA processing genome-wide

2016; Nature Portfolio; Volume: 11; Issue: 3 Linguagem: Inglês

10.1038/nprot.2016.012

1754-2189

Takayuki Nojima, Tomás Gomes, Maria Carmo‐Fonseca, Nicholas Proudfoot,

RNA and protein synthesis mechanisms

mNET-seq generates genome-wide, single-nucleotide–resolution data on Pol II occupancy and co-transcriptional RNA processing, with the unique ability to link these processes to Pol II C-terminal domain phosphorylation states. The transcription cycle of RNA polymerase II (Pol II) correlates with changes to the phosphorylation state of its large subunit C-terminal domain (CTD). We recently developed Native Elongation Transcript sequencing using mammalian cells (mNET-seq), which generates single-nucleotide–resolution genome-wide profiles of nascent RNA and co-transcriptional RNA processing that are associated with different CTD phosphorylation states. Here we provide a detailed protocol for mNET-seq. First, Pol II elongation complexes are isolated with specific phospho-CTD antibodies from chromatin solubilized by micrococcal nuclease digestion. Next, RNA derived from within the Pol II complex is size fractionated and Illumina sequenced. Using mNET-seq, we have previously shown that Pol II pauses at both ends of protein-coding genes but with different CTD phosphorylation patterns, and we have also detected phosphorylation at serine 5 (Ser5-P) CTD-specific splicing intermediates and Pol II accumulation over co-transcriptionally spliced exons. With moderate biochemical and bioinformatic skills, mNET-seq can be completed in ∼6 d, not including sequencing and data analysis.