Extended Data Fig. 3: BANC-seq derived \(K_d^{Apps}\) comparison with other methods.
(a) Spike-in normalized sequencing reads per site and titration point of FLAG-SP1 in MCF-7 relative to the highest signal for the BBC3 and KLF3 regulatory element (dotted line indicating the \(K_d^{Apps}\)). (b) Top: EMSA of 1 nM biotinylated dsDNA binding to recombinant FLAG-SP1 in a concentration range between 5-4000 nM for the BBC3 and KLF3 regulatory element. The experiments were performed thrice with similar results, and one representative image was chosen for visualization. Bottom: Quantification of immunoblotting results, with FLAG-SP1 concentration shown in the logarithmic scale and the bound fraction determined by the ImageJ software. Data are presented as mean ± s.d. c) Overview of detected \(K_d^{Apps}\) by the different methods for the sites depicted in (a). (d) Relative quantification by PAQMAN of SP1 binding in MCF-7 nuclear lysate to the same sequences as in (b). Data are presented as mean ± SEM of two experiments (n = 2). (e) Venn diagram depicting the overlapping and unique proteins that bind to the tested sequences from (d) with high affinity in PAQMAN. (f) Spike-in normalized sequencing reads per site and titration point of FLAG-SP1 in MCF-7 relative to the highest signal for the MEMO1 and RNF223 regulatory element (dotted line indicating the \(K_d^{Apps}\)). (g) Top: EMSA of 1 nM biotinylated dsDNA binding to recombinant FLAG-SP1 in a concentration range between 5-4000 nM for the MEMO1 and RNF223 regulatory element. The experiments were performed thrice with similar results, and one representative image was chosen for visualization. Bottom: Quantification of immunoblotting results, with FLAG-SP1 concentration shown in the logarithmic scale and the bound fraction determined by the ImageJ software. Data are presented as mean ± s.d. (h) Overview of detected \(K_d^{Apps}\) by the different methods for the sites depicted in (f).
