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GreenCUT&RUN: a rapid and efficient genome profiling approach for transcription and chromatin regulatory proteins

ABSTRACT: Chromatin modifiers, transcription factors and cofactors regulate gene expression through engagement with chromatin and DNA, which can be detected by a variety of genomic techniques. Here, we describe a novel method ‘greenCUT&RUN’ for genome-wide profiling of transcription regulatory factors, which has higher sensitivity, resolution, accuracy and reproducibility than existing methods, whilst assuring specificity as assessed for the gene specific transcription factors NFY and c-FOS, and for the basal transcription factor TBP. Our strategy begins with tagging the protein of interest with GFP and utilizes a GFP-specific nanobody fused to MNase to profile genome-wide binding events. Since the nanobody targets GFP, the greenCUT&RUN approach eliminates dependency on protein-specific antibodies, which makes it widely applicable. To examine, if GFP-tagged proteins form complex with known interactors and GFP did not interfere in the formation of protein complex, mass spectrometry was performed.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hela Cell

SUBMITTER: Marc Timmers

LAB HEAD: H. Th. Marc Timmers

PROVIDER: PXD021089 | Pride | 2021-01-13

REPOSITORIES: Pride

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