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Since the episomal system proved to be suitable for the analysis of composite CTCF/TR binding sites, we generated enhancer blocking episomes with the newly identified composite elements.
To better localize the binding sites, we generated heatmaps and graphs of reads smaller than 19 nucleotides.
For multiple adjacent binding sites, we generated the tags for each single binding site, and accumulated the tag counts at all positions from all binding sites.
To analyze the specificity of presence of AP-1 and SP1 binding sites, we generated a set of 100 random 1 kb sequences, which were analyzed in the same way, as our primary dataset.
To identity STAT6 binding sites, we generated a total of 19 099 520 and 18 981 052 36-bp sequence ChIP Seq tags in Ramos and BEAS2B cells, respectively (Tables 1– 3).
To test the sensitivity and specificity of PhyloScan when seeking binding sites that are weaker than E. coli Crp binding sites, we generated "1/2-strength" and "1/3-strength" Crp sites.
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To test our proposed location for the Lrp5/6 binding site, we generated the disease-associated Norrin mutant R121W (Arg121 is a mutational hotspot; Figure 2 figure supplement 2).
To clarify the impact of the second modulator binding site, we generated dimerization curves for the simplified linear models with fixed parameters.
According to a published method [ 9], for each binding site, we generated as many negative site-ligand pairs as the known positive pairs by combining the site with randomly chosen ligands among the other sites' ligands (excluding those known to interact with the given target).
Because several of these point mutations flank or fall within the SH3 binding sites, we also generated a GFP-Lpd 35A 850−1250aa GFP-Lpd 35A 850−1250aawhich lactinonly the bindingesidues that sit between Ena/VASP and Abi1/endophilin binding sites.
To further demonstrate PDA's utility in studying systems biology and structure-based transcription factor binding site prediction, we generated a non-redundant dataset with protein-DNA complex structures that are involved in gene regulation.
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