Protocol for multiplexed transcription factor activity detection using optimized barcoded reporters and an automated computational pipeline

Transcription factors (TFs) regulate the genome in response to signaling events. Detecting their activity is crucial to deciphering the regulatory networks of cells. Here, we present a protocol for multiplexed TF activity detection using a barcoded plasmid library of optimized "prime" TF reporters in cultured cells. We describe steps for library transfection, RNA processing for barcode sequencing, and a computational pipeline for analyzing differential TF activity, enabling high-throughput and quantitative TF profiling. For complete details on the use and execution of this protocol, please refer to Trauernicht et al.¹.