Integrating quantitative proteomics with accurate genome profiling of transcription factors by greenCUT&RUN release_flzgrdhhsjdsthmfzwgc7g3evi

Abstract

<jats:title>Abstract</jats:title> Genome-wide localization of chromatin and transcription regulators can be detected by a variety of techniques. Here, we describe a novel method 'greenCUT&amp;RUN' for genome-wide profiling of transcription regulators, which has a very high sensitivity, resolution, accuracy and reproducibility, whilst assuring specificity. Our strategy begins with tagging of the protein of interest with GFP and utilizes a GFP-specific nanobody fused to MNase to profile genome-wide binding events. By using a GFP-nanobody the greenCUT&amp;RUN approach eliminates antibody dependency and variability. Robust genomic profiles were obtained with greenCUT&amp;RUN, which are accurate and unbiased towards open chromatin. By integrating greenCUT&amp;RUN with nanobody-based affinity purification mass spectrometry, 'piggy-back' DNA binding events can be identified on a genomic scale. The unique design of greenCUT&amp;RUN grants target protein flexibility and yields high resolution footprints. In addition, greenCUT&amp;RUN allows rapid profiling of mutants of chromatin and transcription proteins. In conclusion, greenCUT&amp;RUN is a widely applicable and versatile genome-mapping technique.
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