PEACh: Purifying Elongation complex with Associated factors from Chromatin v1

Here we provide a protocol for purification of RNA Polymerase II elongation complex with associated factors from native chromatin. To isolate high-quality nuclei from mammalian cell culture, adherent cells are lysed directly on a cell culture plate and nuclei are separated from cytoplasmic and other fractions by centrifugation and sucrose cushion. Nuclei can be stored in a freezer. Following chromatin solubilization via enzymatic digestion of nucleic acids, RNA Polymerase II and interacting proteins are enriched using antibody-conjugated magnetic beads. An elution fraction can be used for western blot and proteomic analysis. This method is particularly useful when combined with rapid depletion systems (e.g. auxin-inducible degron/AID, dTAG, PROTAC) to study in vivo functions of transcription elongation factors, including SPT5, SPT6, PAF1, and NELF.