Nojima Takayuki, Gomes Tomás, Carmo-Fonseca Maria, Proudfoot Nicholas J
Sir William Dunn School of Pathology, University of Oxford, Oxford, UK.
Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal.
Nat Protoc. 2016 Mar;11(3):413-28. doi: 10.1038/nprot.2016.012. Epub 2016 Feb 4.
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.
RNA聚合酶II（Pol II）的转录循环与其大亚基C末端结构域（CTD）磷酸化状态的变化相关。我们最近开发了使用哺乳动物细胞的天然延伸转录本测序（mNET-seq），它能生成与不同CTD磷酸化状态相关的全基因组范围的单核苷酸分辨率新生RNA和共转录RNA加工图谱。在此，我们提供mNET-seq的详细方案。首先，用特异性磷酸化CTD抗体从经微球菌核酸酶消化溶解的染色质中分离Pol II延伸复合物。接下来，对Pol II复合物内的RNA进行大小分级并进行Illumina测序。使用mNET-seq，我们先前已表明Pol II在蛋白质编码基因的两端停顿，但具有不同的CTD磷酸化模式，并且我们还检测到丝氨酸5（Ser5-P）CTD特异性剪接中间体的磷酸化以及共转录剪接外显子上的Pol II积累。具备中等的生化和生物信息学技能，mNET-seq可以在约6天内完成，不包括测序和数据分析。
