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Summary: Genome-wide mapping of transcription factors (TFs) is critical to understand their functions. In chromatin immunoprecipitation (ChIP)-seq assay, it’s challenging to study recruitment of low-abundant TFs transiently boud to the genome. Here, we present an optimized protocol using ChIP Next-Gen Seq Sepharose (Staph-seq) to efficiently pull down chromatin complexes. The double size selection promotes sensitive capture of genome-wide protein-DNA associations while eliminating potential Staph A contamination, which is a common problem in protocols using Staph A cells.For complete details on the use and execution of this protocol, please refer to Tao et al. (2020).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.