The technique of chromatin immunoprecipitation (ChIP) is a powerful method for identifying in vivo DNA binding sites of transcription factors and for studying chromatin modifications. Unfortunately, the large number of cells needed for the standard ChIP protocol has hindered the analysis of many biologically interesting cell populations that are difficult to obtain in large numbers. New ChIP methods involving the use of carrier chromatin have been developed that allow the one-gene-at-a-time analysis of very small numbers of cells. However, such methods are not useful if the resultant sample will be applied to genomic microarrays or used in ChIP-sequencing assays. Therefore, we have miniaturized the ChIP protocol such that as few as 10,000 cells (without the addition of carrier reagents) can be used to obtain enough sample material to analyze the entire human genome. We demonstrate the reproducibility of this MicroChIP technique using 2.1 million feature high-density oligonucleotide arrays and antibodies to RNA polymerase II and to histone H3 trimethylated on lysine 27 or lysine 9.

Introduction

We and others (1,2,3,4,5) have developed protocols for studying DNA-protein interactions and histone modifications in living cells or tissues (see also www.epigenome-noe.net/research-tools/protocols.php). In particular, the method of chromatin immunoprecipitation (ChIP) has provided many new insights into gene regulation (6,7). Briefly, ChIP involves crosslinking the transcription factors to their DNA binding sites by treatment of cells with formaldehyde and then preparation of chromatin by sonication of the treated cells. An immunoprecipitation is then performed using the crosslinked chromatin, resulting in the collection of all the binding sites in the genome for the factor of interest. This sample can then be analyzed by PCR to study a particular gene(s) or applied to microarrays for analysis of many genes (8,9,10,11,12). The majority of ChIP studies have used large numbers of cells grown in culture. However, even using the standard amount of 10⁷ cells, there is not enough precipitated sample for hybridization to a single microarray. Investigators have solved this problem by pooling ChIP samples (13,14). However, the problem is exacerbated when considering the number of arrays needed to analyze the entire human genome; pooling samples is simply not practical under these conditions. Therefore, most ChIP-chip protocols incorporate an amplification step that converts the small amount of material present in a ChIP sample into the microgram quantities required for hybridization to an array. Using such amplification techniques, investigators can routinely obtain enough sample material from 10⁶ to 10⁷ cells for analysis of the human genome.

We are interested in applying the ChIP technology to study very small numbers of human cells, such as those obtained from tumor biopsies, fractionation of mixed populations by cell sorting, or differentiation of embryonic stem cells. Although one study has been published in which 1000 cells were used for a ChIP assay, the protocol required mixing a small number of mammalian cells with large numbers of Drosophila cells (15). The success of this protocol was demonstrated by analysis of a few promoters using PCR reactions. Unfortunately, the use of carrier chromatin is not appropriate if the ChIP samples are to be analyzed by sequencing (the carrier DNA would constitute most of the sequenced tags) or by hybridization (due to cross-reaction of the carrier DNA to the oligonucleotides on the array). Therefore, we have taken the approach of altering the ChIP-chip assay such that only 10,000 cells (without carrier DNA) are now required. Importantly, enough sample material can be obtained from 10,000 cells to analyze the entire human genome using micro-array technologies.

Materials and Methods

Cells, Tissues, Antibodies, and PCR

Cells used for the miniaturization optimization are HuH7 human hepatocellular carcinoma cells. Cells were grown at 37°C in a humidified 5% CO₂ incubator in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 100 U/mL of penicillin and streptomycin. The cells were crosslinked and chromatin prepared as described in the MicroChIP Protocol (see Supplementary Material available online at www.BioTechniques.com). For the experiments in this study, the chromatin ranged from 0.5 to 3 kb in size, with a median size of ∼1 kb. Antibodies to RNA polymerase II (8WG16; Covance, Denver, PA, USA), H3me3K27 (07–449; Upstate, Charlottesville, VA, USA), and H3me3K9 (8898; Abcam, Cambridge, MA, USA) were used in the ChIP assays. Primers used to confirm the binding of factors to the promoter region of the described genes have the following sequence: RNAPII: 5′-agatgaaaccgttgtccaaact-3′, 5′-aggttacggcagtttgtctctc-3′; SOAT: 5′-ccctactttccagggacaca-3′, 5′-acaaatgaacatcggcaaca-3′; EVX1: 5′-ccgggcttcacacttctaaa-3′, 5′-aaaggaaacccgcagctaat-3′. and ZNF44: 5′-ggctttcccacacaattcac-3′, 5′-aacactcccgccagagtaga-3′; ZNF333: 5′-cacaggagagaagccctacg-3′, 5′-tcgcgcactcatacagtttc-3′. PCR conditions were 95°C for 3 min, 30 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 45 s, and then 72°C for 5 min.

MicroChIP Protocol (For 10⁴–10⁵ Cells)

This entire protocol can be performed in ∼4 days, assuming that the Staph A cells are prepared in advance (indicated as Day 0). A brief summary of the protocol is provided here; further details, including a step-by-step protocol, catalog numbers for reagents, and composition of each solution is provided in the MicroChIP Protocol (see Supplementary Material).