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Chip-Seq on the Solid™ System

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Chip-Seq on the Solid™ System APPLICATION NOTE ChIP-Seq on the SOLiD™ System Chromatin Immunoprecipitation-based Sequencing (ChIP-Seq) on the SOLiD™ System Introduction Chromatin immunoprecipitation (ChIP) is a technique for identifying and characterizing elements in protein-DNA interactions involved in gene regulation 1. Cross-link bound 2. Isolate chromatin and 3. Precipitate 4. Reverse cross-link 5. Ligate P1 and P2 or chromatin organization. Historically, proteins to DNA. shear DNA. chromatin with and digest protein. adaptors to construct protein-specific fragment library. ChIP reactions were analyzed by PCR, antibody. sequencing and more recently by microarray technologies also known ™ as ChIP-on-chip. Microarray platforms Figure 1: DNA enrichment by chromatin immunoprecipitation(ChIP) and SOLiD fragment library construction. provide a method for “global” ChIP analysis but direct sequencing of SOLiD System ChIP-Seq Analysis precipitated with an antibody that is enriched fragments has proven more The SOLiD System’s ability to generate specific to the DNA-binding protein. effective in determining locations of over 400 million sequence tags (35 bp The quality of this antibody is critical to sequence reads) in a single run enables the success of ChIP-Seq protocols, as it DNA-binding proteins along the genome whole genome ChIP analysis of complex determines the level of enrichment over in an unbiased manner. The massively organisms. Sequence tags are mapped background that is obtained. The DNA is parallel sequencing capacity, high to a reference sequence and counted, released by reversing the cross-link to ™ accuracy and flexibility of the SOLiD to identify specific regions of protein the protein and the protein is digested. System make it well suited for binding. The ultra high throughput of the The size and concentration of the ChIP-Seq applications. system provides researchers with the resulting ChIP DNA fragments deter- sensitivity and the statistical resolving mines the approach that is taken to powers required to map and accurately process this sample for SOLiD fragment characterize the protein-DNA interactions library construction and subsequent of an entire genome. Additionally, sequencing. (Figure 2, page 2) the flexible slide format allows Typically, DNA derived from the ChIP researchers to analyze multiple procedure can range from 100 bp to 2 kb experimental samples and a control in size and is often limiting in quantity. sample in a single run. Therefore, modifications to the standard ChIP analysis with the SOLiD System SOLiD System 2.0 Fragment Library (Figure 1) begins with a traditional Preparation: Lower Input DNA protocol is chromatin immunoprecipitation used to create the ChIP-Seq library. procedure. DNA is cross-linked in vivo to Preparation of a negative control DNA-binding proteins with formaldehyde consisting of non-immunoprecipitated and mechanically sheared using sonica- fragmented DNA of similar size range tion. The DNA-protein complex is then or chromatin-immunoprecipitated TaBLE 1. Comparison OF CHIP Detection Platforms DNA using unspecific IgG antiserum is suggested to detect differential enrich- Feature SOLiD System (ChIP-Seq) Microarray (ChIP-chip) ment. Once these SOLiD ChIP-Seq and Resolution > 400 million sequence tags ~ 6.5 million oligonucleotides negative control libraries are created, the per run per array samples are sequenced on the Genome Coverage UNLIMITED: Entire genome can LIMITED by probe design SOLiD™ System. The short sequence be sequenced hypothesis-free reads from the SOLiD System are map- Specificity No cross-hybridization risks. Cross-hybridization risks between ped against genomic sequences, using Identify unique sequence tags closely related elements the SOLiD System alignment tools avail- Sample Multiplexing YES NO able through the Applied Biosystems Software Development Community (http://info.appliedbiosystems.com/solid- softwarecommunity) or third-party tools ChIP Input DNA Size Range compatible with color space. Data can then be visualized with a tool such as the University of California, Santa Cruz Genome Browser (http://genome.ucsc. >200 bp edu/cgi-bin/hgGateway) to identify and quantify the regions of sequence that bind to the protein of interest. Shear DNA <200 bp SOLiD System ChIP-Seq Analysis of FOXA3 protein In collaboration with the laboratory of End Repair End Repair Dr. Claes Wadelius at Uppsala University, ChIP-Seq analysis was performed using ChIP DNA isolated from hepatic cell lines Ligate Adaptors Ligate Adaptors to identify loci involved in interactions with the FOXA3/HNF3g protein. This hepatocyte nuclear factor, a member 6% PAGE Gel Purify and 6% PAGE Gel Purify and of the forkhead class of DNA-binding Size Selection (150-200 bp) Size Selection (150-200 bp) proteins, activates transcription for liver-specific genes such as albumin and APOA2 and also interacts with DNA and Nick Translation and Nick Translation and Large-Scale PCR Large-Scale PCR histones as a pioneer factor that opens chromatin during development. Materials and Methods: 3% or 4% Agarose Gel-Purify 3% or 4% Agarose Gel-Purify and ChIP DNA was prepared as previously and Size Selection (150-200 bp) Size Selection (150-200 bp) described (Rada-Iglesias et al, 2005) from HepG2 cells using a commercially- Bioanalyzer QC and FlashGel® Bioanalyzer QC and FlashGel® available antibody (Santa Cruz Biotech- for purity and quantitation for purity and quantitation nology; sc-5361) specific to the FOXA3/ HNF3g protein. Immunoprecipitated DNA (0.5 μg) was subjected to size selection and purification using gel Figure 2. ChIP-Seq SOLiD™ fragment library preparation based on ChIP input DNA size ranges. electrophoresis to isolate DNA fragments of 3 different size ranges: as the negative control sample. P1 and generation for each library was 150-200 bp, 200-250 bp, and 250-300 P2 adaptor ligation and all subsequent performed according to SOLiD System bp. Sheared, non-immunoprecipitated steps were performed on all 4 samples 2.0 User Guide standard protocols. Each hepatic cell line DNA (4 μg), similarly according to SOLiD System 2.0 sample was deposited on a quadrant of subjected to size selection and purifica- Fragment Library Preparation: Lower the slide at a target bead density of tion of 250-300 bp fragments, served Input DNA protocol. Templated bead 60-70 K beads/panel. A duplicate slide was generated and 1.) A cut-off was calculated for the over- Results processed similarly to assess the lap signal using the binomial distribution As expected with an enriched sample, reproducibility of the system. as a model under the hypothesis that the the resulting uniquely mapped reads reads were randomly placed onto the covered about 10% of the human High throughput sequencing was genome. genome on average (Table 1). The performed using the SOLiD™ System number of reads with a unique starting and analysis of 35 bp reads was car- 2.) FOXA3 peaks also showing signifi- point averaged approximately 1.07 for ried out. All reads were filtered for high cant signal in the control sample were all samples, indicating that the sample quality reads (0, 1, 2, or 3 mismatches filtered from the analysis. had unique genomic representation and in color space), as well as for alignment 3.) A peak was required to have a signifi- minimal amplification bias. Based on and unique placement in the human cant signal of forward reads upstream these data, we estimate that 5-10 million reference sequence. or a reverse signal downstream from its uniquely mapped reads is sufficient to In these experiments, the data from one center (an example is shown in Figure 3). map all protein-binding regions in a quadrant generated more than enough Putative FOXA3 binding regions were complex genome such as human. reads to map all signals in the genome. visualized using the UCSC Genome All sequence information was used We elected to make use of all the data Browser (http://genome.ucsc.edu). based on the high correlation values by first verifying a high correlation Detection of putative FOXA3 binding between different replicates, ranging between the replicates and then merg- sequences in the enriched peak regions from 0.70-0.83. For peak determination, ing the reads. The reads were extended was done using the BCRANK package a cut off value of 21 overlapping in silico to represent the selected ChIP in R/Bioconductor (http://www.biocon fragments was used at a Bonferroni fragment sizes (bp). The following ductor.org/packages/release/bioc/html/ corrected p-value <0.01 based on our process was used to qualify peaks: BCRANK.html). simulations. Using these stringent chr1: 159459500 159460000 159460500 159461000 FOXA3 binding sequence FOXA3 binding sequence FOXA3 reads 110 _ FOXA3 signal FOXA3 signal 0 _ Input reads 10 _ Input signal Control signal 0 _ UCSC Genes Based on RefSeq, UniProt, GenBank, CCDS and Comparative Genomics APOA2 APOA2 Figure 3. Evidence for FOXA3 protein-DNA interaction at the BCAS1 locus binding to the APOA2 promoter. Graphic representation of alignment of reads derived from size selected SOLiD™ ChIP-Seq libraries (red) and from control input sample (blue) along Chromosome 20 is shown. Illustrated in the UCSC Genome Browser are the mapped forward (red) and reverse (blue) reads and the region of overlap (black). The peak is just upstream of the first exon located and spans the predicted FOXA3 binding motif. criteria, > 4,000 peaks for FOXA3 were (Figure 4), which closely resembles the Conclusion detected either at promoters of protein previously characterized FOXA3 bind- The SOLiD™ System provides a level of coding genes or at a distance from a ing sequence. Interestingly, this motif throughput and sensitivity that cannot gene that is consistent with FOXA3 was found to be centered at the peak be achieved with current hybridization interaction with cis-regulatory elements. maxima, including that of the APOA2 technologies or other next-generation An example of a FOXA3 peak detected promoter. sequencing platforms. The SOLiD by our peak detection strategy is System’s ability to generate over 400 illustrated in Figure 3 using the UCSC million sequence tags, to provide a large Genome Browser.
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