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Knowledge of the transcription factor binding landscape (TFBL) is necessary to analyze gene regulatory networks for important agronomic traits. However, a low‐cost and high‐throughput in vivo chromatin profiling method is still lacking in plants. Here, we developed a transient and simplified cleavage under targets and tagmentation (tsCUT&Tag) that combines transient expression of transcription factor proteins in protoplasts with a simplified CUT&Tag without nucleus extraction. Our tsCUT&Tag method provided higher data quality and signal resolution with lower sequencing depth compared with traditional ChIP‐seq. Furthermore, we developed a strategy combining tsCUT&Tag with machine learning, which has great potential for profiling the TFBL across plant development.

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