Measuring transcription factor binding and gene expression using barcoded self-reporting transposon calling cards and transcriptomes

Calling cards technology using self-reporting transposons enables the identification of DNA-protein interactions through RNA sequencing . Here, we have drastically reduced the cost and labor requirements of calling card experiments in bulk populations of cells by introducing a DNA barcode into the calling card itself. An additional barcode incorporated during reverse transcription enables simultaneous transcriptome measurement in a facile and affordable protocol. We demonstrate that barcoded self-reporting transposons recover in vitro binding sites for four basic helix-loop-helix transcription factors with important roles in cell fate specification: ASCL1, MYOD1, NEUROD2, and NGN1. Further, simultaneous calling cards and transcriptional profiling during transcription factor overexpression identified both binding sites and gene expression changes for two of these factors. In sum, RNA-based identification of transcription factor binding sites and gene expression through barcoded self-reporting transposon calling cards and transcriptomes is an efficient and powerful method to infer gene regulatory networks in a population of cells. We performed mutagenesis of the terminal repeat region of the piggyBac transposon to identify sites that could accommodate or serve as a barcode for self-reporting transposon calling card expeirments. We identified 4 mutable nucleotides and used these sites as barcodes. We constructed barcoded self-reporting transposons encoding either puromycin resistance or tdTomato. We then used these barcoded constructs to identify binding sites for four transcription factors involved in cell fate decisions.