2. Academic Validation
  3. Protocol for multiplexed transcription factor activity detection using optimized barcoded reporters and an automated computational pipeline

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

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: h.yucel@nki.nl.
  • 3 Oncode Institute, Division of Molecular Genetics, Netherlands Cancer Institute, 1066 CX Amsterdam, the Netherlands. Electronic address: b.v.steensel@nki.nl.
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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