Chromatin immunoprecipitation sequencing (ChIP-seq) maps in vivo transcription factor (TF) occupancy under native chromatin conditions. Here, we present a protocol for genome-wide profiling of intrinsic TF motifs using protein-DNA pull-down sequencing (PD-seq), a complementary in vitro technique based on DNA affinity purification sequencing (DAP-seq) with optimizations for mammalian systems. We describe steps for TF purification, naked genomic DNA preparation, protein-DNA pull-down, and sequencing-based analysis. Applied to FOXP3, PD-seq identifies T n G microsatellite repeats as a preferred motif. The protocol bypasses antibody dependence and applies to any affinity-tagged DNA-binding protein. For complete details on the use and execution of this protocol, please refer to Zhang et al. 1 • Instructions for recombinant TF expression and affinity purification • Steps for preparing fragmented naked genomic DNA for pull-down • Guidance on protein-DNA pull-down and sequencing library construction • Procedures for bioinformatic analysis and de novo motif discovery Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Chromatin immunoprecipitation sequencing (ChIP-seq) maps in vivo transcription factor (TF) occupancy under native chromatin conditions. Here, we present a protocol for genome-wide profiling of intrinsic TF motifs using protein-DNA pull-down sequencing (PD-seq), a complementary in vitro technique based on DNA affinity purification sequencing (DAP-seq) with optimizations for mammalian systems. We describe steps for TF purification, naked genomic DNA preparation, protein-DNA pull-down, and sequencing-based analysis. Applied to FOXP3, PD-seq identifies T n G microsatellite repeats as a preferred motif. The protocol bypasses antibody dependence and applies to any affinity-tagged DNA-binding protein.
Jiang et al. (Tue,) studied this question.