Calling Cards is a platform technology to record a cumulative history of transient protein-DNA interactions in the genome of genetically targeted cell types. The record of these interactions is recovered by next-generation sequencing. Compared with other genomic assays, readouts of which provide a snapshot at the time of harvest, Calling Cards enables correlation of historical molecular states to eventual outcomes or phenotypes. To achieve this, Calling Cards uses the piggyBac transposase to insert self-reporting transposon “Calling Cards” into the genome, leaving permanent marks at interaction sites. Calling Cards can be deployed in a variety of in vitro and in vivo biological systems to study gene regulatory networks involved in development, aging, and disease. Out of the box, it assesses enhancer usage but can be adapted to profile-specific transcription factor (TF) binding with custom TF-piggyBac fusion proteins. The Calling Cards workflow has five main stages: delivery of Calling Cards reagents, sample preparation, library preparation, sequencing, and data analysis. Here, we first present a comprehensive guide for experimental design, reagent selection, and optional customization of the platform to study additional TFs. Then, we provide an updated protocol for the five steps, using reagents that improve throughput and decrease costs, including an overview of a newly deployed computational pipeline. This protocol is designed for users with basic molecular biology experience to process samples into sequencing libraries in 2 days. Familiarity with bioinformatic analysis and command line tools is required to set up the pipeline in a high-performance computing environment and to conduct downstream analyses.

Original languageEnglish
Article numbere883
JournalCurrent Protocols
Issue number9
StatePublished - Sep 2023


  • calling cards
  • enhancer usage
  • epigenetics
  • gene regulation
  • transcription factor binding


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