ChIP-seq analysis of protein–DNA interactions on the Ion Proton System

Recent publication highlights usefulness of the Ion Proton System^™ for ChIP-Seq applications with low input material

Epigenetic researcher and Scientific Director at National Jewish Hospital, Dr. Brian P. O’Connor recently teamed with the Thermo Fisher Informatics Applications Group to test Chromatin immunoprecipitation sequencing (ChIP-seq) analysis of protein–DNA interactions on the Ion Proton^™ System.

Advantages of ChIP-Seq over prior ChIP methods

ChIP-seq is the evolution of an earlier microarray method (ChIP-chip) to enrich and map binding sites for transcription factors, chromatin-modifying complexes, histone modifications, and other chromatin-associated proteins.

Massively parallel sequencing of the enriched DNA provides an unbiased, genome-wide view of the binding sites for a diversity of chromatin-associated proteins in any cell type or organism with known genomic sequence. A robust ChIP-seq methodology compatible with small populations of flow cytometry–sorted, highly purified cells is required to accurately define epigenetic and chromatin dynamics regulating cell functions that when disregulated may contribute to disease.

Of key interest in the study of the epigenome are changes that influence chromatin structure and accessibility such as DNA methylation and histone modifications. Global, unbiased methods, such as ChIP-seq, have been crucial to revealing the epigenome and were employed by the ENCODE (Encyclopedia of DNA Elements) project.

Challenges of ChIP with low starting amounts

The isolation of DNA by ChIP typically yields low amounts of starting material that can span a wide size range for next generation sequencing (NGS) library construction. This can be particularly acute when ChIP is performed on rare or limited populations of cells. This research application of semiconductor-based ChIP-seq analysis using sub-nanogram amounts of DNA has been previously demonstrated for the Ion PGM^™ System.

The preliminary data suggests that the longer read lengths possible through semiconductor-based sequencing may be advantageous for ChIP-seq applications and also suggests the scalability of Ion Torrent^™ instruments may enable the optimization of ChIP-seq for limited starting populations for research of specific cell types.

Results of ChIP-Seq using the Ion Torrent ^TM Proton ^TM PI Chip

Dr. O’Connor and the team followed an optimized ChIP-seq workflow. Multiplexing four samples on an Ion PI^™ v2 Chip, replicate runs resulted in excellent barcode balance per sample using the TMAP alignment tool with default parameters.

Genomic binding sites identified using the MAGnify^™ Chromatin Immunoprecipitation System and the Ion Proton^™ System were compared to ENCODE project results for the same cell type. For transcription factor CTCF, a majority of peaks were concordant with an ENCODE dataset, while a smaller, but still significant number were unique to ChIP-seq data generated on the Ion Proton^™ System. Binding sites for histone modifications H3K27ac and H3K27me3 were highly correlated with ENCODE project data.

ChIP-seq analysis was also used to examine the genomic programming that occurs during lineage development of dendritic cells (DCs), whichorchestrate a range of important functions during innate and adaptive immune responses.  Depending on the sample source, cell lines versus in vivo–derived cells, ChIP-seq experiments can be confounded by low-input DNA amounts. To address the challenges of limited sample input and to test the robustness of the optimized ChIP-seq protocol, cell sorted populations of CD11b+ lineage DCs were analyzed for genomic binding sites of the transcription factor Irf4. The Venn diagram below illustrates excellent agreement with significant peaks common across all cell inputs.

In addition, sample multiplexing of four barcoded libraries on a single Ion PI^™ Chip, when compared to ENCODE project data, demonstrated that results for 1 million cells are comparable to those for 20 million cells, thereby enabling epigenomic analysis of limited cell populations.

Conclusions

Specifically developed for research with precious samples such as primary cells, stem cells, and archived sample material, the MAGnify^™ Chromatin Immunoprecipitation System, in combination with an optimized ChIP-seq protocol for the Ion Proton^™ System, demonstrates high-quality data from limited cell populations of flow cytometry–sorted mouse CD11b+ DCs.

The Ion Proton^™ System, with scalable chips, rapid sequencing run times, and affordable instrument pricing, enables the implementation of a ChIP-seq solution that is highly reproducible, robust, and accessible for research studies with sample size variability.

Learn More

Access the full App Note at ChIP-seq analysis of protein–DNA interactions on the Ion Proton^™ System.

Follow these links to the Ion Proton^™ System, the first benchtop sequencing system capable of human-scale genome, exome, or transcriptome sequencing in just a few hours, and the MAGnify^™ Chromatin Immunoprecipitation System, which uses magnetic bead capture technology to provide a streamlined, optimized assay for the enrichment of chromatin/protein complexes and DNA recovery.