Figure 1.

Sequential Extraction Assisted-Active TF Identification. (A) Schematic Representation of the Sea-ATI Workflow. Sea-ATI profiles the cistrome models of the active TFs by enriching ligands bound by TFs (in the nuclear extract) from a complex randomized DNA library. Ground powder of plant tissue is first extracted with a salt-free wash buffer to remove cytoplasmic components, then extracted with a high-salt buffer capable of lysing the nuclei to obtain nuclear proteins. After incubating the nuclear extract with the randomized DNA library, the protein-bound ligands are separated from the unbound ligands by EMSA and gel extraction, and PCR amplified. After 3–5 cycles of enrichment, the bound libraries are sequenced and analyzed. (B) The Sequential Extraction Enriches TFs by Removing the Cytosol. Left, log frequencies of all 8-mers are compared between the stem sea-ATI library and the same library shuffled. Right, E-MI analyses (see Methods); signal strength near the bottom of the triangle (hypotenuse) becomes stronger than elsewhere if TF signals are present. Note that TF signals are detected only in the sea-ATI library enriched with the nuclear extract (bottom) but not with the cytoplasmic extract (top). The corresponding motif of an enriched 8-mer (red circle) is indicated.