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

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