Mapping genome-wide transcription factor binding sites in frozen tissues.

Daniel Savic,Jason Gertz,Richard M Myers,Preti Jain,Gregory M Cooper

doi:10.1186/1756-8935-6-30

Abstract

BackgroundGenome-wide maps of transcription factor binding sites in primary tissues can expand our understanding of genome function, transcriptional regulation, and genetic alterations that contribute to disease risk. However, almost all genome-wide studies of transcription factors have been in cell lines, and performing these experiments in tissues has been technically challenging and limited in throughput.ResultsHere we outline a simple strategy for mapping transcription factor binding sites in frozen tissues that utilizes dry pulverization of samples and is scalable for high-throughput analyses. We show that the method leads to accurate and reproducible chromatin immunoprecipitation next-generation sequencing (ChIP-seq) data, and is highly sensitive, identifying high-quality transcription factor binding sites from chromatin corresponding to only 5 mg of liver tissue.ConclusionsThe enhanced reproducibility, robustness, and sensitivity of the dry pulverization method, in addition to the ease of implementation and scalability, makes ChIP-seq in primary tissues a widely accessible assay.

Highlights

Genome-wide maps of transcription factor binding sites in primary tissues can expand our understanding of genome function, transcriptional regulation, and genetic alterations that contribute to disease risk
We find the method to be robust across diverse tissue types and highly reproducible for both a general transcription factor (Rnap2) and sequence-specific factors (Ctcf and Retinoid X receptor α (Rxrα))
Chromatin immunoprecipitation (ChIP)-seq in mouse tissues To perform ChIP-seq in frozen tissues while minimizing tissue handling and cross-contamination concerns, we developed an approach that uses dry pulverization (Figure 1 and Methods)

Summary

Introduction

Genome-wide maps of transcription factor binding sites in primary tissues can expand our understanding of genome function, transcriptional regulation, and genetic alterations that contribute to disease risk. Almost all genome-wide studies of transcription factors have been in cell lines, and performing these experiments in tissues has been technically challenging and limited in throughput. Several influential ChIP-seq studies have been successfully performed in diverse mouse tissues [15,16,17,18], as well as normal [19] and diseased [20] human tissue samples. These investigations have illustrated the power of genomic assays in primary tissues for characterizing basic cellular functions and the genomic hallmarks common to disease states

Methods

Results

Discussion

Conclusion