Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

Summary

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

Abstract

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.

For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.

Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

Introduction

For many years there has been an unmet need to identify the set of genes bound and regulated by a given protein genome wide, in particular those in the transcription factor class.

Odom et al.¹ used chromatin immunoprecipitation (ChIP) combined with promoter microarrays to systematically identify the genes occupied by pre-specified transcriptional regulators in human liver and pancreatic islets. Subsequently, Johnson et al.² developed a large-scale chromatin immunoprecipitation assay based on direct ultra high-throughput DNA sequencing (ChIP-seq) in order to comprehensively map protein-DNA inte....

Protocol

1. Cross-link Chromatin

1. Grow cells in 100x20mm cell culture dishes. The amount of cells can range from 1 to 10 million cells per dish depending on cell type. Approximately 2 million cells is sufficient for one immunoprecipitation.
2. Cross-link cells in 1% Formaldehyde for 10 min at room temperature with occasional rocking.
3. Quench cross-linking by adding a final concentration of 125 mM Glycine and incubate for 5 min at room temperature.
4. Wash cells with 1X Phosphate Buffered Saline (PBS) twice, decant PBS, and then add 0.2 ml of PBS.
5. Harvest cells with a plastic cell scraper into a microcentrifuge tube.
6. Spin do....

Results

Once the chromatin has been sonicated and have been treated with RNase and Proteinase, the samples run on the 2% agarose gel should present a smear with the bulk of the DNA at the desired size. If several different cycles are tested, a gradual decrease in size should be seen as the number of cycles increase (Figure 2).

After completing the immunoprecipitation portion of the protocol the enrichment can either be checked by PCR or real-time PCR. For PCR samples run on an a.......

Discussion

It is now feasible to carry out a genome-wide profile of protein-DNA interactions association using ChIP-seq, as has been very recently demonstrated with other transcription factors^2,3. The key to a successful sequencing outcome is the generation of a high quality chromatin immunoprecipitation DNA template.

Once the DNA template has been generated and ascertained to be adequately enriched, one can then take it in library preparation for subsequent sequencing. For example, one.......

Materials

Name	Company	Catalog Number	Comments
Name of the reagent	Company	Catalogue number
QIAquick PCR Purification Kit	Qiagen	28104
EZ-ChIP Kit	Millipore	17-371
GoTaq Hot Start Polymerase	Promega	M5001
Misonix Sonicator	Qsonica	XL-2000
NanoDrop 1000 Spectrophotometer	Thermo-Scientific
			Positive control primer sequences (TCF7L2-1) Forward- 5'-TCGCCCTGTCAATAATCTCC-3' Reverse- 5'-GCTCACCTCCTGTATCTTCG-3' Negative control primer sequences (CTRL-1) Forward-5'-ATGTGGTGTGGCTGTGATGGGAAC-3' Reverse- 5'-CGAGCAATCGGTAAATAGGTCTGG-3'