Research paper
A streamlined method for rapid and sensitive chromatin immunoprecipitation

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Abstract

We report a streamlined procedure to efficiently carry samples from chromatin to qPCR-compatible DNA in as little as 4 h. We use this streamlined ChIP to quantify histone H3 modifications at active (cad) and repressed (T early alpha) promoters in a Rag1-deficient pro-T cell line after 1–2 h IP. We further show that the protocol readily quantified histone modifications in chromatin from 104 Rag-deficient DN thymocytes. Taken together, these data outline a simple, cost-effective procedure for efficient ChIP analysis.

Introduction

The fast-growing fields of epigenetics and epigenomics have necessitated development of assays that probe interactions between DNA, structural histone proteins, and regulatory transcription factors. One of the primary tools used to unravel the so-called “histone code” has been chromatin immunoprecipitation, or ChIP (Dedon et al., 1991, O'Neill and Turner, 1996, Kuo and Allis, 1999). Indeed, ChIP, coupled with realtime PCR (qPCR) has become the gold standard assay for chromatin organization (Jenuwein and Allis, 2001), and is increasingly used to demonstrate differential transcription factor recruitment to various promoters (Morshead et al., 2003, O'Neill et al., 2006). Despite such widespread use, the complexity, lengthiness, and scale of the standard ChIP protocol, which can take up to 3 days and require ≥ 106 cells per reaction, make it extremely sensitive to experimenter-induced variability and to contamination, and limit its utility for scarce cell populations, such as those found in select compartments of the immune system.

A number of groups have now proposed modifications to the standard ChIP protocol (Nelson et al., 2006, O'Neill et al., 2006, Attema et al., 2007, Dahl and Collas, 2007, Dahl and Collas, 2008). The newer protocols have demonstrate that ChIP is amenable to variations in virtually every aspect of the assay from the size of chromatin input, the time dedicated to immunoprecipitation, washing, elution, and crosslink reversal, to Proteinase K treatment regiments. By replacing agarose or sepharose beads with Protein A- or Protein G-coupled paramagnetic beads, newer approaches minimize the need to preclear input chromatin of antibody-independent bead binding activities. At the same time, the ability to easily and quantitatively capture magnetic bead complexes eliminates the need for centrifugation, which both reduces the time required for each of the many washes in the ChIP protocol, and reduces the potential for sample loss or contamination during wash aspiration.

We have designed a streamlined protocol that incorporates improvements offered by the Q2-ChIP (Dahl and Collas, 2007), FastChIP (Nelson et al., 2006), ChIP-IT Express (ActiveMotif), and miniChIP (Attema et al., 2007) into a simplified format. We find this streamlined protocol is easily mastered, rapid, and highly reproducible. Demonstrating its adaptability to reduced sample size, our streamlined ChIP readily quantitated histone modifications in as few as 104 CD4/CD8 double negative thymocytes harvested from a Rag-2 deficient mouse. Though we find each of the existing ChIP protocols can be highly effective, we propose our streamlined protocol as a simplified approach for those new to ChIP or for higher throughput ChIP screens.

Section snippets

Cells

The RAG1−/−, p53−/− pro-T cell line, P5424, has been previously described (Mombaerts et al., 1995). P5424 cells were cultured at 37 °C/5% CO2 in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 0.01% penicillin/streptomycin, and 50 µM β-mercaptoethanol. Thymii were isolated from 4–8 weeks old Rag2−/− mice, crushed and filtered to yield a single cell suspension, and red blood cells were removed by hypotonic lysis. The mouse studies described here were reviewed and

Synthesis of a streamlined ChIP protocol from existing methods

Although extremely effective, the standard ChIP protocol as described by Kuo and Allis (1999) remains time-intensive and difficult. By contrast, we recently found that multiple modified ChIP protocols including the Q2ChIP (Dahl and Collas, 2007) and ChIP-IT Express (ActiveMotif) were readily mastered by less experienced lab members. Noting the remarkable differences between each of these effective protocols, we sought to synthesize a single method that would minimize tradeoffs for cost, ease of

Discussion

Although enormously powerful, the conventional methodology for ChIP is a somewhat daunting procedure that requires large amounts of chromatin input and stretches across several days. A series of recent studies have shown that individual steps of the standard ChIP protocol can be extensively modified. We have synthesized the various improvements to the conventional ChIP protocol to develop a simplified method that is significantly shorter, uses a minimum amount of costly reagents and equipment,

Disclosures

The authors have no financial conflicts of interest.

Acknowledgements

We gratefully thank Drs Hosni Hassan and Jose Bruno Barcena for use of their Q-PCR equipment.

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This work was supported by a grant from the National Institute of Allergy and Infectious Diseases (R56AI070848-01A1).

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