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

  • STAR Protoc. 2025 Sep 19;6(3):104066. doi: 10.1016/j.xpro.2025.104066.
Max Trauernicht  1 ,  Vinícius H Franceschini-Santos  1 ,  Hatice Yücel  2 ,  Bas van Steensel  3
Affiliations
  • 1. Oncode Institute, Division of Molecular Genetics, Netherlands Cancer Institute, 1066 CX Amsterdam, the Netherlands.
  • 2. Oncode Institute, Division of Molecular Genetics, Netherlands Cancer Institute, 1066 CX Amsterdam, the Netherlands. Electronic address: [email protected].
  • 3. Oncode Institute, Division of Molecular Genetics, Netherlands Cancer Institute, 1066 CX Amsterdam, the Netherlands. Electronic address: [email protected].
PMID: 40934073 DOI: 10.1016/j.xpro.2025.104066
Abstract

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.

Keywords
Bioinformatics; Cell Biology; Cell culture; Gene Expression; Genomics; Molecular Biology; RNAseq; Sequence analysis; Sequencing; Systems biology.
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