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

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

PROVIDER: PRJNA624288 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR11523300_1.fastq.gz	Fastqsanger.gz
	SRR11523300_2.fastq.gz	Fastqsanger.gz
	SRR11523301_1.fastq.gz	Fastqsanger.gz
	SRR11523301_2.fastq.gz	Fastqsanger.gz
	SRR11523302_1.fastq.gz	Fastqsanger.gz

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

Project description: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 use scCC to map the binding of BRD4 and three additional TFs in cultured cells. We then apply this technique to discover bromodomain-dependent cell state dynamics in K562 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.

2020-07-23 | GSE148448 | GEO

Self-Reporting Transposons Enable Simultaneous Readout of Gene Expression and Transcription Factor Binding in Single Cells.

Project description:Cellular heterogeneity confounds in situ assays of transcription factor (TF) binding. Single-cell RNA sequencing (scRNA-seq) deconvolves cell types from gene expression, but no technology links cell identity to TF binding sites (TFBS) in those cell types. We present self-reporting transposons (SRTs) and use them in single-cell calling cards (scCC), a novel assay for simultaneously measuring gene expression and mapping TFBS in single cells. The genomic locations of SRTs are recovered from mRNA, and SRTs deposited by exogenous, TF-transposase fusions can be used to map TFBS. We then present scCC, which map SRTs from scRNA-seq libraries, simultaneously identifying cell types and TFBS in those same cells. We benchmark multiple TFs with this technique. Next, we use scCC to discover BRD4-mediated cell-state transitions in K562 cells. Finally, we map BRD4 binding sites in the mouse cortex at single-cell resolution, establishing a new method for studying TF biology in situ.

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

Project description: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.

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