title: Mammalian NET-seq analysis defines nascent RNA profiles and associated RNA processing genome-wide.
abstract: 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.
authors: Sir William Dunn School of Pathology, University of Oxford, Oxford, UK.
authors: Takayuki
authors: T
authors: Nojima
authors: Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal.
authors: Tomás
authors: T
authors: Gomes
authors: Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal.
authors: Maria
authors: M
authors: Carmo-Fonseca
authors: Sir William Dunn School of Pathology, University of Oxford, Oxford, UK.
authors: Nicholas J
authors: NJ
authors: Proudfoot
mesh_terms: D000818:Animals; D002460:Cell Line; D020869:Gene Expression Profiling / Q000379:methods; D016678:Genome; D006801:Humans; D010766:Phosphorylation; D012313:RNA / Q000235:genetics* / Q000378:metabolism; D012319:RNA Polymerase II / Q000378:metabolism; D012326:RNA Splicing; D017423:Sequence Analysis, RNA / Q000379:methods*; D014158:Transcription, Genetic; D059467:Transcriptome
chemical_list: D012313:RNA; D012319:RNA Polymerase II