ConnecTF: A platform to integrate validated transcription factor-target interactions to build and refine gene regulatory networks
The direct targets of 16 TFs were identified using the TARGET system. Arabidopsis plants were grown on 1/2X MS media and after 10 days protoplasts were generated from root tissue, Cells were transfected with a vector containing the GR-TF fusion and incubated overnight. Each experiment also included a GR-only empty vector control. In the morning, samples were pre-treated with cycloheximide for 20 minutes, before TF entry into the nucleus was induced with dexamethasone. 3 hours after dex treatment samples were FACS sorted into RNA extraction buffer and RNA-seq libraries were generated. Transcriptional response to each TF compared to empty vector was assayed. Overall design: Genome-wide direct regulated targets of 16 TFs were determined by comparison to an empty vector control. Vectors were expressed in protoplasts isolated from root tissue, samples were done in triplicate, and sequenced using the Illumina NextSeq platform.