Self-reporting transposons enable simultaneous readout of gene expression and transcription factor binding in single cells

Abstract

In situ assays of transcription factor (TF) binding are confounded by cellular heterogeneity and represent averaged profiles in complex tissues. Single cell RNA-seq (scRNA-seq) is capable of resolving different cell types based on gene expression profiles, but no technology exists to directly link specific cell types to the binding pattern of TFs in those cell types. Here, we present self-reporting transposons (SRTs) and their use in single cell calling cards (scCC), a novel assay for simultaneously capturing gene expression profiles and mapping TF binding sites in single cells. First, we show how the genomic locations of SRTs can be recovered from mRNA. Next, we demonstrate that SRTs deposited by the piggyBac transposase can be used to map the genome-wide localization of the TFs SP1, through a direct fusion of the two proteins, and BRD4, through its native affinity for piggyBac. We then present the scCC method, which maps SRTs from scRNA-seq libraries, thus enabling concomitant identification of cell types and TF binding sites in those same cells. As a proof-of-concept, we show recovery of cell type-specific BRD4 and SP1 binding sites from cultured cells. Finally, we map Brd4 binding sites in the mouse cortex at single cell resolution, thus establishing a new technique for studying TF biology in situ.

Measuring Transcription Factor Binding and Gene Expression using Barcoded Self-Reporting Transposon Calling Cards and Transcriptomes

“…Calling card libraries were prepared as described with minor modifications 3,4,16 . We performed first-strand reverse transcription reactions in 20 μL total volume using 2 μg of RNA from each sample.…”

“…A recent technical innovation termed the 'selfreporting transposon' (SRT) allows for the facile recovery of calling cards through RNA sequencing (RNA-seq) 3 . RNA-mediated mapping of transposon insertions is more efficient than previous DNA-based protocols 1 , and this protocol enables the simultaneous identification of TFBS and changes in gene expression in single cells 4 . However, in bulk experiments on populations of cells, the RNA-mediated protocol is technically cumbersome, requiring a large number of replicates 4 .…”

“…RNA-mediated mapping of transposon insertions is more efficient than previous DNA-based protocols 1 , and this protocol enables the simultaneous identification of TFBS and changes in gene expression in single cells 4 . However, in bulk experiments on populations of cells, the RNA-mediated protocol is technically cumbersome, requiring a large number of replicates 4 . Here, we present two crucial modifications of the SRT technology and protocol to facilitate its use and to enable joint recording of TFBS and gene expression in populations of cells: barcoded SRTs and barcoded transcriptomes.…”

“…'Original' calculations use the recommended 12 replicates per TF4 . This experiment assayed 3 TFs (unfused hyper piggyBac, ASCL1, and MYOD1).…”

The qBED track: a novel genome browser visualization for point processes

“…Calling card technology is currently undergoing a renaissance. We have recently used calling cards to study TF binding in bulk populations of cells in vivo (Cammack et al, 2020), and we have also combined calling cards with single cell RNA-seq to simultaneously profile cell identity in complex organs and heterogenous disease states (Moudgil et al, 2019). Calling cards has also been used to dissect TF binding in both steady state and dynamic contexts (Mayhew and Mitra, 2014;Shively et al, 2019).…”