2NimbleGen Systems Inc., Madison, WI, USA
A single chromatin immunoprecipitation (ChIP) sample does not provide enough DNA for hybridization to a genomic tiling array. A commonly used technique for amplifying the DNA obtained from ChIP assays is ligation-mediated PCR (LM-PCR). However, using this amplification method, we could not identify Oct4 binding sites on genomic tiling arrays representing 1% of the human genome (ENCODE arrays). In contrast, hybridization of a pool of 10 ChIP samples to the arrays produced reproducible binding patterns and low background signals. However, the pooling method would greatly increase the number of ChIP reactions needed to analyze the entire human genome. Therefore, we have adapted the GenomePlex® whole genome amplification (WGA) method for use in ChIP-chip assays; detailed ChIP and amplification protocols used for these analyses are provided as supplementary material. When applied to ENCODE arrays, the products prepared using this new method resulted in an Oct4 binding pattern similar to that from the pooled Oct4 ChIP samples. Importantly, the signal-to-noise ratio using the GenomePlex WGA method is superior to the LM-PCR amplification method.
The technique of chromatin immunoprecipitation (ChIP) has proven to be a powerful tool, allowing the detection of protein-DNA interactions in living cells. Although this technique was first adapted for use with mammalian cells less than 10 years ago (1,2), it is now the gold standard experiment for the identification of a target gene of a particular transcription factor. Over the last several years, great strides have been made in expanding the use of ChIP from a one gene-at-a-time approach to a global analysis tool through the hybridization of the samples to genomic microarrays (i.e., the ChIP-chip assay). Today, arrays representing promoter regions (3), CpG islands (4,5,6), or entire genomes (7) are used in combination with ChIP to identify binding sites for transcription factors and components of the transcriptional machinery and to define chromatin structure. However, a single ChIP sample does not provide enough DNA for labeling and hybridization to an array. A commonly used technique for amplifying the DNA obtained from ChIP assays is ligation-mediated PCR (LM-PCR). Unfortunately, we have found that this method often produces very high background when samples are analyzed on genomic tiling arrays. In this study, we have compared three ChIP sample preparation methods that differ in the background noise and reproducibility of binding site identification.Materials and Methods Cell Culture
Ntera2 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM glutamine, 100 U/mL of penicillin and streptomycin, and 10% fetal bovine serum (FBS). All cells were incubated at 37°C in a humidified 5% CO2 incubator.ChIP-Chip Assays
ChIP assays (1 × 107 cells/assay) were performed following the protocol provided in the supplementary materials (available online at www.BioTechniques.com with updates at genomics.ucdavis.edu/farnham and genomecenter.ucdavis.edu/expression_analysis). The Oct4 antibody used in this study was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and the rabbit anti-goat immunoglobulin G (IgG) was purchased from MP Biomedicals (Solon, OH, USA). For PCR analysis of the ChIP samples prior to product generation, QIAquick®-purified immunoprecipitates (Qiagen, Valencia, CA, USA) were dissolved in 50 µL water. Standard PCRs using 2 µL immunoprecipitated DNA were performed. PCR products were separated by electrophoresis through 1.5% agarose gels and visualized using ethidium bromide.
Three different preparation methods were used to obtain enough ChIP DNA for application to genomic microarrays. ChIP-chip experiments were performed using two independent cultures of cross-linked Ntera2 cells for each method.Method 1
LM-PCR. For this method, one half of a ChIP sample (from 1 × 107 cells) was used for linker ligation. Amplification of the linker-ligated DNA using LM-PCR is described in detail at genomics.ucdavis. edu/farnham; see also Reference (8).Method 2
Pooling ChIP samples. For this method, 10 individual Oct4 ChIP assays were performed from each of two sets of 1 × 108 cross-linked cells (1 × 107 cells/ChIP assay). ChIP samples were processed separately following the standard protocol, except that after pre-clearing the chromatin with StaphA cells, all 10 ChIP samples were pooled into one tube for the washing steps. Washes and elution of the pooled ChIPs were then carried out as described in the standard protocol.Method 3
Whole genome amplification (WGA). An adaptation of the standard protocol for WGA using the GenomePlex® WGA kit (Sigma-Aldrich, St. Louis, MO, USA) was used. Briefly, the initial random fragmentation step was eliminated, and an entire ChIP sample (from 1 × 107 cells) or 10 ng input chromatin were amplified. This usually provides enough sample for one array. However, if additional product is needed, then a second round of amplification (using 10–20 ng of the first amplification sample) can be performed. A detailed protocol for the WGA method is provided in the supplementary materials.
Biological replicates of LM-PCR products, pooled ChIP samples, and WGA products (a total of six samples) were applied to ENCODE (Encyclopedia of DNA Elements) oligonucleotide arrays (NimbleGen Systems, Madison, WI, USA) containing approximately 380,000 50-mer probes per array, tiled every 38 bp. The regions included on the arrays encompassed the 30 Mb of the repeat masked ENCODE sequences, representing approximately 1% of the human genome. The labeling of DNA samples for ChIP-chip analysis was performed by NimbleGen Systems, Inc. Briefly, each DNA sample (1 µg) was denatured in the presence of 5′ Cy™3- or Cy5-labeled random nonamers (TriLink Biotechnologies, San Diego, CA, USA) and incubated with 100 U (exo-) Klenow fragment (New England Biolabs, Ipswich, MA, USA) and dNTP mixture [6 mM each in TE buffer (10 mM Tris, 1 mM EDTA, pH 7.4; Invitrogen, Carlsbad, CA, USA)] for 2 h at 37°C. Reactions were terminated by addition of 0.5 M EDTA, pH 8.0, precipitated with isopropanol, and resuspended in water. Then, 13 µg Cy5-labeled ChIP sample and 13 µg Cy3-labeled total sample were mixed, dried down, and resuspended in 40 µL hybridization buffer (NimbleGen Systems) plus 1.5 µg human COT1 DNA. After denaturation, hybridization was carried out in a MAUI® (MicroArray User Interface) Hybridization System (BioMicro Systems, Salt Lake City, UT, USA) for 18 h at 42°C at the NimbleGen Service Laboratory. The arrays were washed using wash buffer system (NimbleGen Systems), dried by centrifugation, and scanned at 5 µm resolution using the GenePix® 4000B scanner (Axon Instruments, Union City, CA, USA). Fluorescence intensity raw data were obtained from scanned images of the oligonucleotide tiling arrays using NimbleScan™ 2.0 extraction software (NimbleGen Systems). For each spot on the array, log2-ratios of the Cy5-labeled test sample versus the Cy3-labeled reference sample were calculated. Then, the biweight mean of this log2 ratio was subtracted from each point; this procedure is approximately equivalent to mean-normalization of each channel. Sites bound by Oct4 were identified using the peak calling algorithm described in Bieda et al. (9), with minor modifications (available upon request). The peaks called for both biological replicates of the LM-PCR, pooling, and WGA methods are provided as supplementary material. The array data has been deposited into Gene Expression Omnibus (GEO; series GSE5251).