Chapter 11 ChIP-Seq to Analyze the Binding of Replication Proteins to Chromatin A. Zachary Ostrow, Christopher J. Viggiani, Jennifer G. Aparicio, and Oscar M. Aparicio Abstract Chromatin immunoprecipitation (ChIP) is a widely used method to study interactions between proteins and discrete chromosomal loci in vivo. ChIP was originally developed for in vivo analysis of protein associations with candidate DNA sequences known or suspected to bind the protein of interest. The advent of DNA microarrays enabled the unbiased, genome-scale identification of all DNA sequences enriched by ChIP, providing a genomic map of a protein’s chromatin binding. This method, termed ChIP-chip, is broadly applicable and has been particularly valuable in DNA replication studies to map potential replication origins in Saccharomyces cerevisiae and other organisms based on the specific association of certain replication proteins with these chromosomal elements, which are distributed throughout the genome. More recently, high-throughput sequencing (HTS) technologies have replaced microarrays as the preferred method for genomic analysis of ChIP experiments, and this combination is termed ChIP-Seq. We present a detailed ChIP-Seq protocol for S. cerevisiae that can be adapted for different HTS platforms and for different organisms. We also outline general schemes for data analysis; however, HTS data analyses usually must be tailored specifically for individual studies, depending on the experimental design, data characteristics, and the genome being analyzed. Key words Chromatin immunoprecipitation, DNA replication, ChIP-Seq, High-throughput sequencing analysis
1 Introduction Chromatin immunoprecipitation (ChIP) is a powerful method used to study the interaction of individual proteins with discrete chromosomal loci in vivo [1, 2]. Protein–DNA or protein–chromatin interactions are stabilized by in vivo chemical cross-linking. The cross-linked chromatin is isolated and sheared randomly to generate discrete chromatin fragments of desired size (~0.5 kb), which determines the resolution of the method. The sheared chromatin is subjected to immunoprecipitation with antibody against the protein of interest, thus enriching for associated DNA sequences. Sonya Vengrova and Jacob Dalgaard (eds.), DNA Replication: Methods and Protocols, Methods in Molecular Biology, vol. 1300, DOI 10.1007/978-1-4939-2596-4_11, © Springer Science+Business Media New York 2015
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In basic ChIP, the presence of specific, candidate DNA sequences is typically determined by quantitative (or semiquantitative) PCR amplification of a limited number of potential DNA-binding and nonbinding (control) loci. While this approach can yield great insights, important data may be missed due to the limited number of loci analyzed. The analysis of many sequences by this approach can be time-consuming and expensive and limited by the quantity of experimental sample. The application of whole-genome analysis methods, first DNA microarrays and more recently high-throughput sequencing (HTS), is a powerful advance allowing determination of all DNA sequences enriched in a particular ChIP experiment [3–11]. These methods, referred to as ChIP-chip and ChIP-Seq, respectively, circumvent the need for candidate loci, which may be biased, and provide, at least in principle, a genomic map of chromatin binding sites of the protein of interest (under the given experimental conditions). Both methods essentially involve the conversion of the chromatin immunoprecipitated (ChIPed) DNA, through the addition of primer adapters, into a DNA “library” that may be amplified by PCR for analysis by microarray or HTS. Because the sequencing approach provides a relative count of the number of times each immunoprecipitated DNA sequence is present versus the relative hybridization intensities measured by the microarray approach, ChIP-Seq is more quantitative and data analysis more straightforward than for ChIPchip, and the resolution is not limited by the microarray design, although it may be limited by the number of sequencing reads in relation to genome size (see Note 1). For these reasons and as sequencing costs have decreased, ChIP-Seq has become the generally preferred method for analysis. In the first edition of this book, we described a detailed protocol for ChIP-chip of budding yeast proteins [12]. Here we present an updated version of that protocol in which the microarray analysis has been replaced by HTS. ChIP-Seq potentially can be applied to any chromatin-associated protein for which an effective antibody is available, or by expressing an epitope-tagged version of the protein of interest. Subheadings 3.1– 3.4 essentially recapitulate and update a previously described ChIP protocol [2]. The methods given for cross-linking and chromatin extraction in Subheadings 3.1 and 3.2 are for the budding yeast, Saccharomyces cerevisiae. Alternative methods for other organisms may be substituted for these sections. Subheadings 3.3 and 3.4 describe immunoprecipitation and DNA purification is generally applicable to various experimental systems and may be modified as required. In Subheading 3.5 samples are prepared for the selected sequencing platform with the use of library preparation kits or custom protocols. In Subheading 3.6 an outline of standard procedures for quality control of a ChIP-Seq library is provided. In Subheading 3.7 the fundamentals of preprocessing sequencing reads are provided, and a model pipeline is presented both to transform raw sequence reads into binned reads, allowing subsequent
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genomic analyses, and to call differentially bound loci across samples. Whereas a basic level of statistical analysis may be sufficient to provide a useful view of the data, deriving more quantitative or subtle information will likely require a more sophisticated, customized approach. For more detailed methods for data analysis, we refer the reader to several publications and external links.
2 Materials Use Milli-Q water to prepare all stock solutions. 2.1 Cross-Linking and Harvesting Cells
1. Yeast extract-peptone-dextrose growth media (YEPD): 2 % (w/v) Bacto peptone, 1 % (w/v) yeast extract, 2 % (w/v) dextrose. 2. 37 % formaldehyde. 3. 2.5 M glycine, autoclaved. 4. Tris-buffered saline (TBS): 100 mM Tris–HCl, pH 7.6, 150 mM NaCl, autoclaved. 5. 50 mL screw-cap tubes.
2.2 Cell Lysis, Chromatin Fragmentation, and Isolation
1. ChIP lysis buffer: 50 mM HEPES–KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 1 % Triton X-100, 0.1 % (w/v) sodium deoxycholate. Filter sterilize and store at 4 °C. 2. Roche Complete Protease Inhibitor Cocktail Tablet, Mini. Custom protease inhibitor cocktails may be substituted. 3. Glass beads, 425–600 μm in diameter, washed and autoclaved. 4. MP Biomedicals FastPrep FP120. Other vortexers or cell disruptors also may be used (see Note 2). 5. 2 mL microcentrifuge tube with gasket-sealed screw caps (required for FastPrep) (e.g., VWR). 6. 26-G × 1/2 in. hypodermic needles. 7. 5 mL polypropylene snap-cap tubes. 8. Covaris S2 Sonicator. Other sonicators may be used (see Note 3). 9. Covaris 12 × 24 mm glass screw-cap tubes.
2.3 Immunopre cipitation
1. Microcentrifuge tube rotator or agitator (e.g., Nutator, LaqQuake). 2. Protein G-sepharose beads in a 50:50 slurry in phosphate- buffered saline (PBS) and 0.01 % (w/v) sodium azide (optional as a preservative). A working solution of PBS is 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, and 1.4 mM KH2PO4 and is typically made as a 10× stock and diluted into distilled water as needed. To prepare the Protein G beads (which are often stored
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in ethanol as a preservative), wash beads three times by l ow-speed centrifugation (350 × g) using ten bead volumes of ice- cold PBS. After the final wash, resuspend the beads in a volume of ice-cold PBS containing 0.01 % sodium azide equal to the bead volume, and store at 4 °C for up to several months. Use of wide-bore pipette tips is recommended when pipetting beads. 3. Lysis buffer-500: 50 mM HEPES–KOH, pH 7.5, 500 mM NaCl, 1 mM EDTA, 1 % Triton X-100, 0.1 % (w/v) sodium deoxycholate. Filter sterilize. 4. LiCl-detergent wash buffer: 10 mM Tris–HCl, pH 8.0, 0.25 M LiCl, 0.5 % Triton X-100, 0.5 % (w/v) sodium deoxycholate, 1 mM EDTA. Filter sterilize. 5. TE: 10 mM Tris–HCl, pH 7.6, 1 mM EDTA. Heat sterilize. 6. Elution buffer: 10 mM Tris–HCl, pH 7.6, 1 mM EDTA, 0.5 % (w/v) SDS. 2.4 DNA Purification
1. 20 mg/mL DNase-free RNase A. Store at −20 °C. 2. Proteinase K solution: 20 mg/mL Proteinase K in 50 mM Tris–HCl, pH 7.6, 1 mM CaCl2. Store at −20 °C. 3. Qiagen MinElute PCR purification kit which includes buffers PB, PE, and EB. Similar DNA purification columns from other vendors that enable the sample to be eluted into a small volume (~10 μL) may be suitable but have not been tested. Prepare 0.2× buffer EB for a modified elution protocol.
2.5 DNA Library Preparation
1. Library preparation kit (e.g., Illumina ChIP-Seq DNA Sample Prep Kit) or custom library preparation reagents dependent upon desired sequencing platform [13].
2.6 Quality Control of Libraries
1. qPCR machine for determination of DNA concentration.
2.7 Processing of Sequencing Reads and Analysis
1. Access to a high-throughput sequencing instrument, most commonly through a dedicated facility.
2. Access to Agilent Technologies BioAnalyzer.
2. Sequencing analysis software (e.g., Bowtie2, SAMtools, BEDTools, MACS, DiffBind).
3 Methods 3.1 Cross-Linking and Harvesting Cells
1. For each sample, grow 200 mL of yeast cells to OD600 ~1.0 (~5 × 109 total haploid cells) in YEPD media. 2. Cross-link the chromatin: To the 200 mL culture, add 5.6 mL of 37 % formaldehyde solution (1 % final concentration), mix gently, and incubate for 15 min at room temperature, with occasional mixing (see Note 4).
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3. Quench the cross-linking: Add 10 mL of 2.5 M glycine (125 mM final concentration) to the cross-linked culture and mix gently. Incubate at room temperature for 5 min. 4. Harvest the cells: For each sample, split cultures into four 50 mL screw-cap tubes. Pellet the cells by centrifugation in swinging-bucket rotor at 1,500 × g for 5 min at 4 °C. Discard the supernatant as hazardous waste. Place the tube containing the cell pellet on ice. 5. Wash: Resuspend each cell pellet by pipetting up and down in 10 mL of ice-cold TBS and pool into two 50-mL screw-cap tubes. Pellet the cells by centrifugation at 1,500 × g for 5 min at 4 °C. Discard the supernatant and place the tube with cell pellet on ice. At this point, each 200 mL culture will have been divided evenly into two sample tubes. 6. Wash each pellet with 20 mL of ice-cold TBS and centrifuge at 1,500 × g for 5 min at 4 °C. Discard the supernatant and place the tubes with cell pellets on ice. 7. Resuspend each cell pellet in 1 mL of ice-cold TBS with a pipetman and transfer each to a separate 2-mL FastPrep microcentrifuge tube on ice. Pellet the cells using a microcentrifuge at full speed (~16,000 × g) for a few seconds. Remove the supernatant without disturbing the cell pellet. At this point the cell pellets may be flash frozen using a dry ice–ethanol or liquid nitrogen bath and stored at −80 °C. There should be two equal cell pellets for each 200 mL culture. 3.2 Cell Lysis, Chromatin Fragmentation, and Isolation
1. Thaw/resuspend the cells: If cells were frozen, thaw on ice. Resuspend each cell pellet in 500 μL of ice-cold ChIP lysis buffer containing 1× protease inhibitors (one protease inhibitor tablet per 10 mL ChIP lysis buffer). 2. Lyse the cells: Add an equal volume (~0.6 mL) of glass beads to the cell suspensions (use a 0.6-mL microcentrifuge tube to measure and dispense beads). 3. Place the tightly capped tubes into a FastPrep in a 4 °C cold room, and run at power setting 5.5 for 45 s (see Note 2). Remove the tubes from the FastPrep and spin in a microcentrifuge at full speed for a few seconds to collapse any foam; place the tubes on ice for ~2 min. 4. Repeat Subheading 3.2, step 3. 5. Separate the lysate from the beads: Wipe the tube bottom with a Kimwipe to remove ice or water droplets. Invert the tube, and flick the tube to knock the beads and solution away from the bottom of the tube. Puncture the inverted tube’s bottom twice with a red-hot 26-G needle (use a small syringe to hold the needle). Immediately insert the tube (it may only fit partially) into a 5-mL polypropylene snap-cap tube (cap removed)
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on ice and centrifuge in a prechilled, swinging-bucket rotor at 350 × g for 2 min at 4 °C to collect the lysate. Remove the tubes from the centrifuge and place on ice. 6. The lysate is partially pelleted at this point. Resuspend the pellet into the soluble phase and transfer the entire lysate to a prechilled fresh tube for sonication (Covaris 12 × 24 mm tube) on ice. 7. Shear the chromatin: Using a Covaris S2 sonicator, fragment chromatin (duty cycle 20 %, intensity 5, cycles/burst 200, mode: frequency sweeping, for eight cycles at 30 s per cycle). Place tubes on ice after sonication, and transfer sonicated, solubilized chromatin to a fresh prechilled microcentrifuge tube, combining the split samples. At this point, the entire chromatin sample from 200 mL cells is contained in one microcentrifuge tube. An alternative sonicator may be used if desired (see Note 5). 8. Remove the cell debris: Centrifuge the samples at full speed for 5 min at 4 °C to pellet the cell debris. Decant the supernatant into a fresh, prechilled microcentrifuge tube. 9. Centrifuge the samples at full speed for 15 min at 4 °C. Decant the supernatant into a fresh, prechilled microcentrifuge tube. This material (~1 mL) is the fragmented chromatin. At this point, the DNA shear size may be determined (see Note 3). 3.3 Immunopre cipitation
1. Optional (see Note 6). Preclear the chromatin extract: Add preimmune serum or nonspecific antibody from a similar source to the fragmented chromatin, and with a wide-bore pipette tip, add 30 μL of a 50:50 suspension of Protein G-sepharose beads and incubate at 4 °C for 1 h with rotation or gentle agitation. Gently pellet the beads using a microcentrifuge at ~800 × g for 1 min at 4 °C. Transfer supernatant (~1 mL) into a new microcentrifuge tube on ice. 2. Immunoprecipitate: Add the appropriate amount of antibody (see Note 7) to the fragmented chromatin. Incubate for at least 2 h (up to overnight) with rotation or gentle agitation at 4 °C. 3. Using a wide-bore pipette tip, add 100 μL of a 50:50 suspension of Protein G-sepharose beads and incubate at 4 °C for 1 h with rotation or gentle agitation. Magnetic beads (e.g., DynaBeads) can be used in place of sepharose beads (see Note 8). 4. Gently pellet the beads using a microcentrifuge at ~800 × g for 1 min at room temperature. Remove supernatant, carefully avoiding the beads; it is better to leave a small volume of supernatant as this will be diluted subsequently, rather than to disturb the beads. Retain supernatant for analysis of DNA shear size if desired (see Note 3).
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5. Wash the beads: Add 1 mL of ChIP lysis buffer (without protease inhibitors) and rotate or gently agitate for ~5 min at room temperature. Gently pellet the beads and carefully remove the supernatant as in Subheading 3.3, step 4. 6. Repeat Subheading 3.3, step 5. 7. Repeat Subheading 3.3, step 5 using 1 mL of lysis buffer-500. 8. Repeat Subheading 3.3, step 5 using 1 mL of LiCl wash buffer. 9. Repeat Subheading 3.3, step 5 using 1 mL of TE. Carefully remove as much supernatant as possible without disturbing beads. 10. Elute the immunoprecipitate: Add 100 μL of elution buffer to the beads and incubate at 65 °C for 10 min in a heat block to elute precipitate from the Protein G-sepharose beads. 11. Pellet the beads in a microcentrifuge at full speed for a few seconds. Remove the eluate (100 μL) and transfer to a fresh microcentrifuge tube. 12. Reverse the cross-links: Incubate the immunoprecipitated (IP) chromatin samples at 65 °C for at least 6 h (up to overnight) (an air incubator is recommended to minimize sample evaporation-condensation inside the tube). 3.4 DNA Purification
1. After cross-link reversal, add 1 μL of RNase A (20 μg total) and incubate for 15 min at 37 °C. 2. Add 5 μL of Proteinase K (100 μg total) and incubate for 1 h at 42 °C. 3. Using a MinElute PCR purification kit and microcentrifuge for Subheading 3.4, steps 3–7, add 500 μL of buffer PB to each sample and mix. Load each sample into a MinElute column and centrifuge for 1 min at full speed. Discard the flow through. 4. Add 750 μL of buffer PE into the column and centrifuge for 1 min at full speed. Discard the flow through. 5. Centrifuge the MinElute column for 1 min at full speed to remove residual buffer PE. 6. Place each column into a fresh microcentrifuge tube. Add 11 μL of 0.2× buffer EB directly to the filter surface of the column and incubate at room temperature for 1 min. 7. Centrifuge each column for 1 min at full speed to elute the DNA; ~10 μL will be recovered. Samples may be stored overnight at 4 °C or a few weeks at −20 °C; for longer-term storage, dry the DNA samples before storing at −20 or −80 °C.
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8. At this point one may proceed to the library preparation steps. However, before investing time and resources into these steps, it is advisable to confirm the expected level of enrichment (if possible) by direct PCR analysis (see Note 9). 3.5 DNA Library Preparation
ChIPed DNA is converted into a DNA library to enable its amplification by PCR to generate sufficient DNA for HTS. Processing involves a series of steps to repair the DNA ends and ligate primer adapters to the DNA ends and PCR amplification. These procedures and materials, particularly the primer adapter sequences, are specific to the different HTS platforms (e.g., Illumina, SOLiD, 454), so libraries must be constructed as specified for the desired platform. Kits for library preparations may be purchased directly from vendors (e.g., ChIP-Seq DNA Sample Prep Kit (Illumina), NEB). Alternatively, detailed protocols outlining the preparation of libraries using commonly available reagents may be more cost effective and are amenable to customization [13]. Core facilities often offer library preparation services, which may be cost effective. Library preparations typically require multiple DNA purification steps. We recommend the use of Agencourt AMPure XP beads for these purifications. This reagent improves DNA recovery compared with column purification methods, and the size range of recovered DNA can be manipulated by varying the relative amount of beads to the DNA used.
3.6 Quality Control of Libraries
Library concentrations and size distributions should be determined before sequencing. These measurements are used to adjust libraries to the concentration required by the particular sequencing instrument. These services are often provided by a sequencing core but can be performed in-house if the proper instruments are available. 1. Library concentrations should be calculated with qPCR. The concentrations of ChIP libraries after amplification should be in excess of that required by the sequencing platform to be used (e.g., 10 μL of 10 nM is ample for Illumina Hi-Seq); Nanodrop and Qubit are usually not accurate or precise enough for this purpose. 2. Library size distribution should be assessed using a BioAnalyzer to ensure that fragments are in the expected size range (see Note 10) and that undesired nucleic acid by-products such as primer dimers have been removed.
3.7 Processing of Sequencing Reads and Analysis
After a sequencing run is complete, raw sequence reads are aligned to the genome. There are many programs available (e.g., Bowtie2 [14]) for this purpose. This step is performed using a terminal or a graphic user interface (GUI) such as Galaxy [15]. We recommend the former for greater control of the processing, as analytic
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options are available that have not been imported into the GUI environment. For installation and execution of the preferred alignment program, the program documentation should be consulted. Aligned reads are imported directly into analytical programs (e.g., MACS, Galaxy [16]) for various analyses such as differential peak calling and visualization of chromosomal plots or further processed and binned using a combination of programs such as SAMtools and BEDTools or related toolsets [17, 18]. A model pipeline to convert sequencing reads into an aligned, binned dataset is as follows [19]: 1. Align reads with Bowtie 2. There are many options available to fine-tune the alignment, for example, altering the permitted number of mismatches between a read and the genome, varying settings to alter the speed of alignment, and adjusting settings specific for paired-end or single-end reads such as varying the range of base pairs allowed between paired-end reads. 2. Filter out multiply aligned reads with SAMtools view. It is important to filter out multiply aligned reads to avoid artificial enrichment of repetitive regions, for example, at origins found in rDNA repeats or near transposable elements. This command can also be used to convert between .sam and .bam formats. 3. To bin reads, create a .bed file of uniform genomic intervals of a desired size using BEDTools makewindows using a reference file of chromosome number and size. 4. Bin aligned reads using BEDTools coverageBed using the read file and the interval file from Subheading 3.7, steps 2 and 3. This data may now be used to create chromosome plots, analyze signal around specific features (e.g., replication origins), and perform other customized analyses. 5. Filtered, aligned reads generated in Subheading 3.7, step 2, may be analyzed directly with a peak caller such as Modelbased Analysis for ChIP-Seq (MACS) to determine the amount and position of peaks. For statistical analysis and peak calling, experimental replicates are required. 6. Data from Subheading 3.7, step 5, can be analyzed for differential binding using a tool such as DiffBind [20] software package designed for the R environment. Various other approaches are available for statistical analysis of the data, depending on the experimental design, data quality, availability of data replicates, etc. Data should be normalized across experiments and appropriately smoothed in a preferred coding environment. Chromosomal data can be analyzed and visualized genome-wide through many techniques, for example, creation of a two-dimensional binary matrix in which columns are centered on a particular genomic feature and rows are representative of specific instances of that feature [21].
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4 Notes 1. ChIP-Seq data are measured in “counts”, i.e., the number of times a base at a given genomic position or bin is present in the sequencing results. This is determined by summing aligned base sequence reads for each genome position. Because sequencing depth is important for the quality of downstream analysis and is inversely proportional to genome size, the number of reads obtained from a sequencing run is limiting for resolution and dynamic range. Thus, the number of reads required for adequate genome coverage should be taken into consideration. Depending on the output of the sequencing instrument and the coverage required, it may be possible to multiplex samples in a single sequencing run to maximize value. Multiplexing typically requires “indexing” or “barcoding” different samples during the library preparation and must be customized to the HTS platform [13]. 2. Cell breakage using the FastPrep is highly efficient, reproducible, and rapid. Standard vortexers may be used; however, multivortexers that can process many microcentrifuge tubes together while operating continuously for several minutes are preferable. For cell breakage, these devices typically require at least 5 min of constant vortexing at the maximum power setting but can vary considerably in their efficacy. Samples should be vortexed in a 4 °C cold room and may be chilled on ice periodically as needed between extended vortexing periods. Using 2 mL microcentrifuge or FastPrep tubes (which have a nearly flat bottom versus the conical shape of standard 1.7 mL microcentrifuge tubes) may improve cell lysis by allowing better agitation of the beads. Assess cell breakage by examining cells under a light microscope. Take 2 μL of cell suspension before breakage (Subheading 3.2, step 1) and after different intervals of cell breakage (Subheading 3.2, step 3); raise the volume to 10–20 μL with water and place on microscope slide with cover slip. With the FastPrep, we typically achieve >95 % breakage; with a Dade multi-vortexer we achieve about 50–70 % breakage. 3. Sonication is a critical step because it solubilizes the chromatin and determines the length of the DNA fragments in the chromatin that will be immunoprecipitated and, hence, affects the potential resolution of the procedure. In general, the smaller the shear size, the better the potential resolution of binding site identification. Sonication does not effectively shear DNA below ~200 bp in length. Oversonication may damage chromatin and protein epitopes, diminishing experimental efficiency. Other approaches for fragmenting DNA to smaller sizes are feasible such as micrococcal nuclease digestion [22]. Sonicator settings
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must be determined if a different sonicator is used, and in any case, the DNA shear size is an important parameter that should be determined for a particular instrument. The number of rounds of sonication may be varied to modulate the DNA shear size. To determine the DNA shear size, the cross-links must be reversed and the DNA analyzed by 2 % agarose gel electrophoresis. Visualization with EtBr requires that about 20 % of the total sample be run on the gel. Usually, a fraction of the total chromatin remaining after immunoprecipitation may be sacrificed for this purpose. Take ~100 μL of fragmented chromatin (after Subheading 3.3, step 4, if immunoprecipitation will be performed) and add 300 μL of elution buffer. Proceed with Subheading 3.3, step 12, transfer sample to fresh 1.7-mL microfuge tube, and purify the DNA by phenol/chloroform/ isoamyl alcohol (25:24:1) extraction and ethanol precipitation (this purification method is used because of the sample volume and amount of protein in the samples). Analyze the DNA on a 2 % agarose gel; it should appear as a smear with the majority of DNA in the 200–1,000 bp range. 4. In principle, varying the time or temperature of cross-linking may improve results by better stabilizing in vivo associations. However, we have not found that increasing the length of cross-linking has any significant effect on ChIP efficiency of ORC (OMA, unpublished). On the contrary, extensive cross- linking (e.g., overnight) can make cell breakage more difficult and may damage epitopes. Increased temperature (e.g., 37 °C) can raise the level of background but may be necessary in certain cases (e.g., working with temperature-sensitive strains). 5. With the Branson 250 sonicator (using microtip attachment), with the microtip horn submerged about halfway down the depth of the solution in the microcentrifuge tube, sonicate for 12 s with constant output on low power. Always keep the horn tip submerged while sonicating. Use power setting 1.5 and 100 % duty cycle (see Note 3). After sonication, place the sample on ice for at least 2 min. Repeat this step at least twice, more times if smaller DNA shear size is desired. 6. Preclearing of the extract is intended to reduce nonspecific immunoprecipitation of chromatin or DNA. We have not observed a significant effect of preclearing in standard ChIP analysis with anti-HA monoclonal antibody 12CA5 but have not tested its effect in ChIP-Seq. As there is no anticipated harmful effect of preclearing (other than consumption of reagents), this step is recommended. 7. As with any procedure involving immunoprecipitation, the quality of antibody-target protein interaction is critical to achieving success. Antibody should be in excess over target protein. Conditions for specific antibodies, such as lysis buffer
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composition and wash buffer stringencies, may need to be adjusted. In addition to using specific monoclonal and polyclonal antibodies raised against proteins such as ORC and MCM subunits, we have also had success with anti-HA, anti- Myc, and anti-Flag antibodies against the corresponding epitope-tagged proteins. We strongly recommend determining the optimal antibody concentration that maximizes signal to noise in a standard ChIP experiment (by direct PCR analysis of binding and nonbinding sites). This is done by aliquoting the chromatin extracts (up to ten aliquots) from Subheading 3.2, step 9, and incubating aliquots with different amounts of antibody, before proceeding with Subheading 3.3, step 3. An alternative approach, which does not depend on prior success of ChIP and knowledge of binding sites, is to determine the minimum amount of antibody that effectively immunoprecipitates the maximum amount of target protein from the chromatin extract. Aliquots of the chromatin extract are subject to immunoprecipitation with different antibody amounts as earlier, and the procedure is continued through Subheading 3.3, step 9. The immunoprecipitates are eluted by addition of ~30 μL of 2× SDS-PAGE sample buffer and incubated at 95 °C for 30 min (this is necessary to reverse the cross-links), followed by Western blot analysis. It is expected that the amount of coprecipitated target protein will reach a maximum once a saturating amount of antibody is reached, which is a reasonable starting point for these experiments. It also may be useful to analyze depletion of the target protein from the extract; however, we have found that complete depletion of the extract often does not occur, possibly because cross-linking damages or obscures epitopes. 8. The use of magnetic beads (e.g., DynaBeads, Life T echnologies) is recommended for ease of application, which may reduce variability and increase recovery in the immunoprecipitation procedure. Additionally, protocols for the cross-linking of antibodies to DynaBeads are available. 9. It is generally anticipated that ChIP-Seq will be performed with proteins that previously have been successfully ChIPed and analyzed by direct, quantitative (or semiquantitative) PCR analysis of known binding and nonbinding sites. The efficacy of the current experiment can be similarly tested before proceeding. We recommend diluting 1 μL of the IP DNA (from Subheading 3.4, step 7) with 39 μL of TE and using 1 μL of this dilution for each analytical PCR reaction. The exact PCR conditions used will be based on methods previously established in the individual laboratory. 10. When assessing the size distribution of libraries, it is important to consider that ligation of primer adapters during library preparation increases the size distribution of the library by the
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total length of the adapters (e.g., a total increase of ~80 bp in preparations for Illumina Hi-Seq). Moreover, primers used in the amplification step of library preparation can form dimers which can be observed in BioAnalyzer traces. Care must be taken to remove as much primer dimer as reasonably possible as they will be sequenced if present in the final library, thus reducing the total number of useful reads.
Acknowledgments We thank J. Dalton for the assistance in establishing lab sequencing protocols. The work was supported by NIH grants 5R01-GM065494 (to O.M.A.), P50-HG002790 (A.Z.O.), and P30CA014089 from the National Cancer Institute (to USC Norris Cancer Center) and by NSF-MRI award #0923513 (to S. Nuzhdin) and by a pilot grant from the USC Epigenome Center sponsored by the Whittier Foundation (to O.M.A.). References 1. Hecht A, Grunstein M (1999) Mapping DNA interaction sites of chromosomal proteins using immunoprecipitation and polymerase chain reaction. Methods Enzymol 304: 399–414 2. Aparicio OM (1999) In: Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (eds) Current protocols in molecular biology. Immunoprecipitation for determining the association of proteins with specific genomic sequences In Vivo John Wiley and Sons Inc, New York. pp 21.3. 1–21.3.12 3. Hayashi M, Katou Y, Itoh T, Tazumi A, Yamada Y, Takahashi T, Nakagawa T, Shirahige K, Masukata H (2007) Genome-wide localization of pre-RC sites and identification of replication origins in fission yeast. EMBO J 26: 1327–1339 4. Xu W, Aparicio JG, Aparicio OM, Tavare S (2006) Genome-wide mapping of ORC and Mcm2p binding sites on tiling arrays and identification of essential ARS consensus sequences in S. cerevisiae. BMC Genomics 7:276 5. Wyrick JJ, Aparicio JG, Chen T, Barnett JD, Jennings EG, Young RA, Bell SP, Aparicio OM (2001) Genome-wide distribution of ORC and MCM proteins in S. cerevisiae: high- resolution mapping of replication origins. Science 294:2357–2360 6. MacAlpine DM, Rodriguez HK, Bell SP (2004) Coordination of replication and
t ranscription along a Drosophila chromosome. Genes Dev 18:3094–3105 7. Bermejo R, Doksani Y, Capra T, Katou YM, Tanaka H, Shirahige K, Foiani M (2007) Top1- and Top2-mediated topological transitions at replication forks ensure fork progression and stability and prevent DNA damage checkpoint activation. Genes Dev 21: 1921–1936 8. Katou Y, Kanoh Y, Bando M, Noguchi H, Tanaka H, Ashikari T, Sugimoto K, Shirahige K (2003) S-phase checkpoint proteins Tof1 and Mrc1 form a stable replication-pausing complex. Nature 424:1078–1083 9. Ren B, Robert F, Wyrick JJ, Aparicio O, Jennings EG, Simon I, Zeitlinger J, Schreiber J, Hannett N, Kanin E, Volkert TL, Wilson CJ, Bell SP, Young RA (2000) Genome-wide location and function of DNA-binding proteins. Science 290:2306–2309 10. Barski A, Cuddapah S, Cui K, Roh T-Y, Schones DE, Wang Z, Wei G, Chepelev I, Zhao K (2007) High-resolution profiling of histone methylations in the human genome. Cell 129:823–837 11. Dellino GI, Cittaro D, Piccioni R, Luzi L, Banfi S, Segalla S, Cesaroni M, Mendoza- Maldonado R, Giacca M, Pelicci PG (2012) Genome-wide mapping of human DNA- replication origins: levels of transcription at ORC1 sites regulate origin selection and replication timing. Genome Res 23:1–11
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12. Viggiani CJ, Aparicio JG, Aparicio OM (2009) ChIP-chip to analyze the binding of replication proteins to chromatin using oligonucleotide DNA microarrays. Methods Mol Biol 521:255–278 13. Dunham JP, Friesen ML (2013) A cost- effective method for high-throughput construction of illumina sequencing libraries. Cold Spring Harb Protoc 2013:820–834 14. Langmead B, Salzberg S (2012) Fast gapped- read alignment with Bowtie 2. Nat Methods 9:357–359 15. Goecks J, Nekrutenko A, Taylor J, The GalaxyTeam (2010) Galaxy: a comprehensive approach for supporting accessible, reproducible, and transparent computational research in the life sciences. Genome Biol 11:R86 16. Zhang Y, Liu T, Meyer CA, Eeckhoute J, Johnson DS, Bernstein BE, Nusbaum C, Myers RM, Brown M, Li W, Liu XS (2008) Model- based analysis of ChIP–seq (MACS). Genome Biol 9:R137 17. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R (2009) The sequence alignment/
map format and SAMtools. Bioinformatics 25:2078–2079 18. Quinlan AR, Hall IM (2010) BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26:841–842 19. Peace JM, Ter-Zakarian A, Aparicio OM (2014) Rif1 regulates initiation timing of late replication origins throughout the S. cerevisiae genome. PLoS ONE 9:e98501. doi:10.1371/ journal.pone.0098501 20. Stark R, Brown GD (2011) DiffBind: differential binding analysis of ChIP-seq peak data. Bioconductor http://bioconductor.org/packages/release/bioc/html/DiffBind.html 21. Ostrow AZ, Nellimoottil T, Knott SRV, Fox CA, Tavaré S et al (2014) Fkh1 and Fkh2 bind multiple chromosomal elements in the S. cerevisiae genome with distinct specificities and cell cycle dynamics. PLoS ONE 9(2):e87647. doi:10.1371/journal.pone. 0087647 22. Yuan GC, Liu YJ, Dion MF, Slack MD, Wu LF, Altschuler SJ, Rando OJ (2005) Genomescale identification of nucleosome positions in S. cerevisiae. Science 309:626–630
1 Introduction Chromatin immunoprecipitation (ChIP) is a powerful method used to study the interaction of individual proteins with discrete chromosomal loci in vivo [1, 2]. Protein–DNA or protein–chromatin interactions are stabilized by in vivo chemical cross-linking. The cross-linked chromatin is isolated and sheared randomly to generate discrete chromatin fragments of desired size (~0.5 kb), which determines the resolution of the method. The sheared chromatin is subjected to immunoprecipitation with antibody against the protein of interest, thus enriching for associated DNA sequences. Sonya Vengrova and Jacob Dalgaard (eds.), DNA Replication: Methods and Protocols, Methods in Molecular Biology, vol. 1300, DOI 10.1007/978-1-4939-2596-4_11, © Springer Science+Business Media New York 2015
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In basic ChIP, the presence of specific, candidate DNA sequences is typically determined by quantitative (or semiquantitative) PCR amplification of a limited number of potential DNA-binding and nonbinding (control) loci. While this approach can yield great insights, important data may be missed due to the limited number of loci analyzed. The analysis of many sequences by this approach can be time-consuming and expensive and limited by the quantity of experimental sample. The application of whole-genome analysis methods, first DNA microarrays and more recently high-throughput sequencing (HTS), is a powerful advance allowing determination of all DNA sequences enriched in a particular ChIP experiment [3–11]. These methods, referred to as ChIP-chip and ChIP-Seq, respectively, circumvent the need for candidate loci, which may be biased, and provide, at least in principle, a genomic map of chromatin binding sites of the protein of interest (under the given experimental conditions). Both methods essentially involve the conversion of the chromatin immunoprecipitated (ChIPed) DNA, through the addition of primer adapters, into a DNA “library” that may be amplified by PCR for analysis by microarray or HTS. Because the sequencing approach provides a relative count of the number of times each immunoprecipitated DNA sequence is present versus the relative hybridization intensities measured by the microarray approach, ChIP-Seq is more quantitative and data analysis more straightforward than for ChIPchip, and the resolution is not limited by the microarray design, although it may be limited by the number of sequencing reads in relation to genome size (see Note 1). For these reasons and as sequencing costs have decreased, ChIP-Seq has become the generally preferred method for analysis. In the first edition of this book, we described a detailed protocol for ChIP-chip of budding yeast proteins [12]. Here we present an updated version of that protocol in which the microarray analysis has been replaced by HTS. ChIP-Seq potentially can be applied to any chromatin-associated protein for which an effective antibody is available, or by expressing an epitope-tagged version of the protein of interest. Subheadings 3.1– 3.4 essentially recapitulate and update a previously described ChIP protocol [2]. The methods given for cross-linking and chromatin extraction in Subheadings 3.1 and 3.2 are for the budding yeast, Saccharomyces cerevisiae. Alternative methods for other organisms may be substituted for these sections. Subheadings 3.3 and 3.4 describe immunoprecipitation and DNA purification is generally applicable to various experimental systems and may be modified as required. In Subheading 3.5 samples are prepared for the selected sequencing platform with the use of library preparation kits or custom protocols. In Subheading 3.6 an outline of standard procedures for quality control of a ChIP-Seq library is provided. In Subheading 3.7 the fundamentals of preprocessing sequencing reads are provided, and a model pipeline is presented both to transform raw sequence reads into binned reads, allowing subsequent
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genomic analyses, and to call differentially bound loci across samples. Whereas a basic level of statistical analysis may be sufficient to provide a useful view of the data, deriving more quantitative or subtle information will likely require a more sophisticated, customized approach. For more detailed methods for data analysis, we refer the reader to several publications and external links.
2 Materials Use Milli-Q water to prepare all stock solutions. 2.1 Cross-Linking and Harvesting Cells
1. Yeast extract-peptone-dextrose growth media (YEPD): 2 % (w/v) Bacto peptone, 1 % (w/v) yeast extract, 2 % (w/v) dextrose. 2. 37 % formaldehyde. 3. 2.5 M glycine, autoclaved. 4. Tris-buffered saline (TBS): 100 mM Tris–HCl, pH 7.6, 150 mM NaCl, autoclaved. 5. 50 mL screw-cap tubes.
2.2 Cell Lysis, Chromatin Fragmentation, and Isolation
1. ChIP lysis buffer: 50 mM HEPES–KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 1 % Triton X-100, 0.1 % (w/v) sodium deoxycholate. Filter sterilize and store at 4 °C. 2. Roche Complete Protease Inhibitor Cocktail Tablet, Mini. Custom protease inhibitor cocktails may be substituted. 3. Glass beads, 425–600 μm in diameter, washed and autoclaved. 4. MP Biomedicals FastPrep FP120. Other vortexers or cell disruptors also may be used (see Note 2). 5. 2 mL microcentrifuge tube with gasket-sealed screw caps (required for FastPrep) (e.g., VWR). 6. 26-G × 1/2 in. hypodermic needles. 7. 5 mL polypropylene snap-cap tubes. 8. Covaris S2 Sonicator. Other sonicators may be used (see Note 3). 9. Covaris 12 × 24 mm glass screw-cap tubes.
2.3 Immunopre cipitation
1. Microcentrifuge tube rotator or agitator (e.g., Nutator, LaqQuake). 2. Protein G-sepharose beads in a 50:50 slurry in phosphate- buffered saline (PBS) and 0.01 % (w/v) sodium azide (optional as a preservative). A working solution of PBS is 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, and 1.4 mM KH2PO4 and is typically made as a 10× stock and diluted into distilled water as needed. To prepare the Protein G beads (which are often stored
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in ethanol as a preservative), wash beads three times by l ow-speed centrifugation (350 × g) using ten bead volumes of ice- cold PBS. After the final wash, resuspend the beads in a volume of ice-cold PBS containing 0.01 % sodium azide equal to the bead volume, and store at 4 °C for up to several months. Use of wide-bore pipette tips is recommended when pipetting beads. 3. Lysis buffer-500: 50 mM HEPES–KOH, pH 7.5, 500 mM NaCl, 1 mM EDTA, 1 % Triton X-100, 0.1 % (w/v) sodium deoxycholate. Filter sterilize. 4. LiCl-detergent wash buffer: 10 mM Tris–HCl, pH 8.0, 0.25 M LiCl, 0.5 % Triton X-100, 0.5 % (w/v) sodium deoxycholate, 1 mM EDTA. Filter sterilize. 5. TE: 10 mM Tris–HCl, pH 7.6, 1 mM EDTA. Heat sterilize. 6. Elution buffer: 10 mM Tris–HCl, pH 7.6, 1 mM EDTA, 0.5 % (w/v) SDS. 2.4 DNA Purification
1. 20 mg/mL DNase-free RNase A. Store at −20 °C. 2. Proteinase K solution: 20 mg/mL Proteinase K in 50 mM Tris–HCl, pH 7.6, 1 mM CaCl2. Store at −20 °C. 3. Qiagen MinElute PCR purification kit which includes buffers PB, PE, and EB. Similar DNA purification columns from other vendors that enable the sample to be eluted into a small volume (~10 μL) may be suitable but have not been tested. Prepare 0.2× buffer EB for a modified elution protocol.
2.5 DNA Library Preparation
1. Library preparation kit (e.g., Illumina ChIP-Seq DNA Sample Prep Kit) or custom library preparation reagents dependent upon desired sequencing platform [13].
2.6 Quality Control of Libraries
1. qPCR machine for determination of DNA concentration.
2.7 Processing of Sequencing Reads and Analysis
1. Access to a high-throughput sequencing instrument, most commonly through a dedicated facility.
2. Access to Agilent Technologies BioAnalyzer.
2. Sequencing analysis software (e.g., Bowtie2, SAMtools, BEDTools, MACS, DiffBind).
3 Methods 3.1 Cross-Linking and Harvesting Cells
1. For each sample, grow 200 mL of yeast cells to OD600 ~1.0 (~5 × 109 total haploid cells) in YEPD media. 2. Cross-link the chromatin: To the 200 mL culture, add 5.6 mL of 37 % formaldehyde solution (1 % final concentration), mix gently, and incubate for 15 min at room temperature, with occasional mixing (see Note 4).
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3. Quench the cross-linking: Add 10 mL of 2.5 M glycine (125 mM final concentration) to the cross-linked culture and mix gently. Incubate at room temperature for 5 min. 4. Harvest the cells: For each sample, split cultures into four 50 mL screw-cap tubes. Pellet the cells by centrifugation in swinging-bucket rotor at 1,500 × g for 5 min at 4 °C. Discard the supernatant as hazardous waste. Place the tube containing the cell pellet on ice. 5. Wash: Resuspend each cell pellet by pipetting up and down in 10 mL of ice-cold TBS and pool into two 50-mL screw-cap tubes. Pellet the cells by centrifugation at 1,500 × g for 5 min at 4 °C. Discard the supernatant and place the tube with cell pellet on ice. At this point, each 200 mL culture will have been divided evenly into two sample tubes. 6. Wash each pellet with 20 mL of ice-cold TBS and centrifuge at 1,500 × g for 5 min at 4 °C. Discard the supernatant and place the tubes with cell pellets on ice. 7. Resuspend each cell pellet in 1 mL of ice-cold TBS with a pipetman and transfer each to a separate 2-mL FastPrep microcentrifuge tube on ice. Pellet the cells using a microcentrifuge at full speed (~16,000 × g) for a few seconds. Remove the supernatant without disturbing the cell pellet. At this point the cell pellets may be flash frozen using a dry ice–ethanol or liquid nitrogen bath and stored at −80 °C. There should be two equal cell pellets for each 200 mL culture. 3.2 Cell Lysis, Chromatin Fragmentation, and Isolation
1. Thaw/resuspend the cells: If cells were frozen, thaw on ice. Resuspend each cell pellet in 500 μL of ice-cold ChIP lysis buffer containing 1× protease inhibitors (one protease inhibitor tablet per 10 mL ChIP lysis buffer). 2. Lyse the cells: Add an equal volume (~0.6 mL) of glass beads to the cell suspensions (use a 0.6-mL microcentrifuge tube to measure and dispense beads). 3. Place the tightly capped tubes into a FastPrep in a 4 °C cold room, and run at power setting 5.5 for 45 s (see Note 2). Remove the tubes from the FastPrep and spin in a microcentrifuge at full speed for a few seconds to collapse any foam; place the tubes on ice for ~2 min. 4. Repeat Subheading 3.2, step 3. 5. Separate the lysate from the beads: Wipe the tube bottom with a Kimwipe to remove ice or water droplets. Invert the tube, and flick the tube to knock the beads and solution away from the bottom of the tube. Puncture the inverted tube’s bottom twice with a red-hot 26-G needle (use a small syringe to hold the needle). Immediately insert the tube (it may only fit partially) into a 5-mL polypropylene snap-cap tube (cap removed)
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on ice and centrifuge in a prechilled, swinging-bucket rotor at 350 × g for 2 min at 4 °C to collect the lysate. Remove the tubes from the centrifuge and place on ice. 6. The lysate is partially pelleted at this point. Resuspend the pellet into the soluble phase and transfer the entire lysate to a prechilled fresh tube for sonication (Covaris 12 × 24 mm tube) on ice. 7. Shear the chromatin: Using a Covaris S2 sonicator, fragment chromatin (duty cycle 20 %, intensity 5, cycles/burst 200, mode: frequency sweeping, for eight cycles at 30 s per cycle). Place tubes on ice after sonication, and transfer sonicated, solubilized chromatin to a fresh prechilled microcentrifuge tube, combining the split samples. At this point, the entire chromatin sample from 200 mL cells is contained in one microcentrifuge tube. An alternative sonicator may be used if desired (see Note 5). 8. Remove the cell debris: Centrifuge the samples at full speed for 5 min at 4 °C to pellet the cell debris. Decant the supernatant into a fresh, prechilled microcentrifuge tube. 9. Centrifuge the samples at full speed for 15 min at 4 °C. Decant the supernatant into a fresh, prechilled microcentrifuge tube. This material (~1 mL) is the fragmented chromatin. At this point, the DNA shear size may be determined (see Note 3). 3.3 Immunopre cipitation
1. Optional (see Note 6). Preclear the chromatin extract: Add preimmune serum or nonspecific antibody from a similar source to the fragmented chromatin, and with a wide-bore pipette tip, add 30 μL of a 50:50 suspension of Protein G-sepharose beads and incubate at 4 °C for 1 h with rotation or gentle agitation. Gently pellet the beads using a microcentrifuge at ~800 × g for 1 min at 4 °C. Transfer supernatant (~1 mL) into a new microcentrifuge tube on ice. 2. Immunoprecipitate: Add the appropriate amount of antibody (see Note 7) to the fragmented chromatin. Incubate for at least 2 h (up to overnight) with rotation or gentle agitation at 4 °C. 3. Using a wide-bore pipette tip, add 100 μL of a 50:50 suspension of Protein G-sepharose beads and incubate at 4 °C for 1 h with rotation or gentle agitation. Magnetic beads (e.g., DynaBeads) can be used in place of sepharose beads (see Note 8). 4. Gently pellet the beads using a microcentrifuge at ~800 × g for 1 min at room temperature. Remove supernatant, carefully avoiding the beads; it is better to leave a small volume of supernatant as this will be diluted subsequently, rather than to disturb the beads. Retain supernatant for analysis of DNA shear size if desired (see Note 3).
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5. Wash the beads: Add 1 mL of ChIP lysis buffer (without protease inhibitors) and rotate or gently agitate for ~5 min at room temperature. Gently pellet the beads and carefully remove the supernatant as in Subheading 3.3, step 4. 6. Repeat Subheading 3.3, step 5. 7. Repeat Subheading 3.3, step 5 using 1 mL of lysis buffer-500. 8. Repeat Subheading 3.3, step 5 using 1 mL of LiCl wash buffer. 9. Repeat Subheading 3.3, step 5 using 1 mL of TE. Carefully remove as much supernatant as possible without disturbing beads. 10. Elute the immunoprecipitate: Add 100 μL of elution buffer to the beads and incubate at 65 °C for 10 min in a heat block to elute precipitate from the Protein G-sepharose beads. 11. Pellet the beads in a microcentrifuge at full speed for a few seconds. Remove the eluate (100 μL) and transfer to a fresh microcentrifuge tube. 12. Reverse the cross-links: Incubate the immunoprecipitated (IP) chromatin samples at 65 °C for at least 6 h (up to overnight) (an air incubator is recommended to minimize sample evaporation-condensation inside the tube). 3.4 DNA Purification
1. After cross-link reversal, add 1 μL of RNase A (20 μg total) and incubate for 15 min at 37 °C. 2. Add 5 μL of Proteinase K (100 μg total) and incubate for 1 h at 42 °C. 3. Using a MinElute PCR purification kit and microcentrifuge for Subheading 3.4, steps 3–7, add 500 μL of buffer PB to each sample and mix. Load each sample into a MinElute column and centrifuge for 1 min at full speed. Discard the flow through. 4. Add 750 μL of buffer PE into the column and centrifuge for 1 min at full speed. Discard the flow through. 5. Centrifuge the MinElute column for 1 min at full speed to remove residual buffer PE. 6. Place each column into a fresh microcentrifuge tube. Add 11 μL of 0.2× buffer EB directly to the filter surface of the column and incubate at room temperature for 1 min. 7. Centrifuge each column for 1 min at full speed to elute the DNA; ~10 μL will be recovered. Samples may be stored overnight at 4 °C or a few weeks at −20 °C; for longer-term storage, dry the DNA samples before storing at −20 or −80 °C.
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8. At this point one may proceed to the library preparation steps. However, before investing time and resources into these steps, it is advisable to confirm the expected level of enrichment (if possible) by direct PCR analysis (see Note 9). 3.5 DNA Library Preparation
ChIPed DNA is converted into a DNA library to enable its amplification by PCR to generate sufficient DNA for HTS. Processing involves a series of steps to repair the DNA ends and ligate primer adapters to the DNA ends and PCR amplification. These procedures and materials, particularly the primer adapter sequences, are specific to the different HTS platforms (e.g., Illumina, SOLiD, 454), so libraries must be constructed as specified for the desired platform. Kits for library preparations may be purchased directly from vendors (e.g., ChIP-Seq DNA Sample Prep Kit (Illumina), NEB). Alternatively, detailed protocols outlining the preparation of libraries using commonly available reagents may be more cost effective and are amenable to customization [13]. Core facilities often offer library preparation services, which may be cost effective. Library preparations typically require multiple DNA purification steps. We recommend the use of Agencourt AMPure XP beads for these purifications. This reagent improves DNA recovery compared with column purification methods, and the size range of recovered DNA can be manipulated by varying the relative amount of beads to the DNA used.
3.6 Quality Control of Libraries
Library concentrations and size distributions should be determined before sequencing. These measurements are used to adjust libraries to the concentration required by the particular sequencing instrument. These services are often provided by a sequencing core but can be performed in-house if the proper instruments are available. 1. Library concentrations should be calculated with qPCR. The concentrations of ChIP libraries after amplification should be in excess of that required by the sequencing platform to be used (e.g., 10 μL of 10 nM is ample for Illumina Hi-Seq); Nanodrop and Qubit are usually not accurate or precise enough for this purpose. 2. Library size distribution should be assessed using a BioAnalyzer to ensure that fragments are in the expected size range (see Note 10) and that undesired nucleic acid by-products such as primer dimers have been removed.
3.7 Processing of Sequencing Reads and Analysis
After a sequencing run is complete, raw sequence reads are aligned to the genome. There are many programs available (e.g., Bowtie2 [14]) for this purpose. This step is performed using a terminal or a graphic user interface (GUI) such as Galaxy [15]. We recommend the former for greater control of the processing, as analytic
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options are available that have not been imported into the GUI environment. For installation and execution of the preferred alignment program, the program documentation should be consulted. Aligned reads are imported directly into analytical programs (e.g., MACS, Galaxy [16]) for various analyses such as differential peak calling and visualization of chromosomal plots or further processed and binned using a combination of programs such as SAMtools and BEDTools or related toolsets [17, 18]. A model pipeline to convert sequencing reads into an aligned, binned dataset is as follows [19]: 1. Align reads with Bowtie 2. There are many options available to fine-tune the alignment, for example, altering the permitted number of mismatches between a read and the genome, varying settings to alter the speed of alignment, and adjusting settings specific for paired-end or single-end reads such as varying the range of base pairs allowed between paired-end reads. 2. Filter out multiply aligned reads with SAMtools view. It is important to filter out multiply aligned reads to avoid artificial enrichment of repetitive regions, for example, at origins found in rDNA repeats or near transposable elements. This command can also be used to convert between .sam and .bam formats. 3. To bin reads, create a .bed file of uniform genomic intervals of a desired size using BEDTools makewindows using a reference file of chromosome number and size. 4. Bin aligned reads using BEDTools coverageBed using the read file and the interval file from Subheading 3.7, steps 2 and 3. This data may now be used to create chromosome plots, analyze signal around specific features (e.g., replication origins), and perform other customized analyses. 5. Filtered, aligned reads generated in Subheading 3.7, step 2, may be analyzed directly with a peak caller such as Modelbased Analysis for ChIP-Seq (MACS) to determine the amount and position of peaks. For statistical analysis and peak calling, experimental replicates are required. 6. Data from Subheading 3.7, step 5, can be analyzed for differential binding using a tool such as DiffBind [20] software package designed for the R environment. Various other approaches are available for statistical analysis of the data, depending on the experimental design, data quality, availability of data replicates, etc. Data should be normalized across experiments and appropriately smoothed in a preferred coding environment. Chromosomal data can be analyzed and visualized genome-wide through many techniques, for example, creation of a two-dimensional binary matrix in which columns are centered on a particular genomic feature and rows are representative of specific instances of that feature [21].
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4 Notes 1. ChIP-Seq data are measured in “counts”, i.e., the number of times a base at a given genomic position or bin is present in the sequencing results. This is determined by summing aligned base sequence reads for each genome position. Because sequencing depth is important for the quality of downstream analysis and is inversely proportional to genome size, the number of reads obtained from a sequencing run is limiting for resolution and dynamic range. Thus, the number of reads required for adequate genome coverage should be taken into consideration. Depending on the output of the sequencing instrument and the coverage required, it may be possible to multiplex samples in a single sequencing run to maximize value. Multiplexing typically requires “indexing” or “barcoding” different samples during the library preparation and must be customized to the HTS platform [13]. 2. Cell breakage using the FastPrep is highly efficient, reproducible, and rapid. Standard vortexers may be used; however, multivortexers that can process many microcentrifuge tubes together while operating continuously for several minutes are preferable. For cell breakage, these devices typically require at least 5 min of constant vortexing at the maximum power setting but can vary considerably in their efficacy. Samples should be vortexed in a 4 °C cold room and may be chilled on ice periodically as needed between extended vortexing periods. Using 2 mL microcentrifuge or FastPrep tubes (which have a nearly flat bottom versus the conical shape of standard 1.7 mL microcentrifuge tubes) may improve cell lysis by allowing better agitation of the beads. Assess cell breakage by examining cells under a light microscope. Take 2 μL of cell suspension before breakage (Subheading 3.2, step 1) and after different intervals of cell breakage (Subheading 3.2, step 3); raise the volume to 10–20 μL with water and place on microscope slide with cover slip. With the FastPrep, we typically achieve >95 % breakage; with a Dade multi-vortexer we achieve about 50–70 % breakage. 3. Sonication is a critical step because it solubilizes the chromatin and determines the length of the DNA fragments in the chromatin that will be immunoprecipitated and, hence, affects the potential resolution of the procedure. In general, the smaller the shear size, the better the potential resolution of binding site identification. Sonication does not effectively shear DNA below ~200 bp in length. Oversonication may damage chromatin and protein epitopes, diminishing experimental efficiency. Other approaches for fragmenting DNA to smaller sizes are feasible such as micrococcal nuclease digestion [22]. Sonicator settings
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must be determined if a different sonicator is used, and in any case, the DNA shear size is an important parameter that should be determined for a particular instrument. The number of rounds of sonication may be varied to modulate the DNA shear size. To determine the DNA shear size, the cross-links must be reversed and the DNA analyzed by 2 % agarose gel electrophoresis. Visualization with EtBr requires that about 20 % of the total sample be run on the gel. Usually, a fraction of the total chromatin remaining after immunoprecipitation may be sacrificed for this purpose. Take ~100 μL of fragmented chromatin (after Subheading 3.3, step 4, if immunoprecipitation will be performed) and add 300 μL of elution buffer. Proceed with Subheading 3.3, step 12, transfer sample to fresh 1.7-mL microfuge tube, and purify the DNA by phenol/chloroform/ isoamyl alcohol (25:24:1) extraction and ethanol precipitation (this purification method is used because of the sample volume and amount of protein in the samples). Analyze the DNA on a 2 % agarose gel; it should appear as a smear with the majority of DNA in the 200–1,000 bp range. 4. In principle, varying the time or temperature of cross-linking may improve results by better stabilizing in vivo associations. However, we have not found that increasing the length of cross-linking has any significant effect on ChIP efficiency of ORC (OMA, unpublished). On the contrary, extensive cross- linking (e.g., overnight) can make cell breakage more difficult and may damage epitopes. Increased temperature (e.g., 37 °C) can raise the level of background but may be necessary in certain cases (e.g., working with temperature-sensitive strains). 5. With the Branson 250 sonicator (using microtip attachment), with the microtip horn submerged about halfway down the depth of the solution in the microcentrifuge tube, sonicate for 12 s with constant output on low power. Always keep the horn tip submerged while sonicating. Use power setting 1.5 and 100 % duty cycle (see Note 3). After sonication, place the sample on ice for at least 2 min. Repeat this step at least twice, more times if smaller DNA shear size is desired. 6. Preclearing of the extract is intended to reduce nonspecific immunoprecipitation of chromatin or DNA. We have not observed a significant effect of preclearing in standard ChIP analysis with anti-HA monoclonal antibody 12CA5 but have not tested its effect in ChIP-Seq. As there is no anticipated harmful effect of preclearing (other than consumption of reagents), this step is recommended. 7. As with any procedure involving immunoprecipitation, the quality of antibody-target protein interaction is critical to achieving success. Antibody should be in excess over target protein. Conditions for specific antibodies, such as lysis buffer
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composition and wash buffer stringencies, may need to be adjusted. In addition to using specific monoclonal and polyclonal antibodies raised against proteins such as ORC and MCM subunits, we have also had success with anti-HA, anti- Myc, and anti-Flag antibodies against the corresponding epitope-tagged proteins. We strongly recommend determining the optimal antibody concentration that maximizes signal to noise in a standard ChIP experiment (by direct PCR analysis of binding and nonbinding sites). This is done by aliquoting the chromatin extracts (up to ten aliquots) from Subheading 3.2, step 9, and incubating aliquots with different amounts of antibody, before proceeding with Subheading 3.3, step 3. An alternative approach, which does not depend on prior success of ChIP and knowledge of binding sites, is to determine the minimum amount of antibody that effectively immunoprecipitates the maximum amount of target protein from the chromatin extract. Aliquots of the chromatin extract are subject to immunoprecipitation with different antibody amounts as earlier, and the procedure is continued through Subheading 3.3, step 9. The immunoprecipitates are eluted by addition of ~30 μL of 2× SDS-PAGE sample buffer and incubated at 95 °C for 30 min (this is necessary to reverse the cross-links), followed by Western blot analysis. It is expected that the amount of coprecipitated target protein will reach a maximum once a saturating amount of antibody is reached, which is a reasonable starting point for these experiments. It also may be useful to analyze depletion of the target protein from the extract; however, we have found that complete depletion of the extract often does not occur, possibly because cross-linking damages or obscures epitopes. 8. The use of magnetic beads (e.g., DynaBeads, Life T echnologies) is recommended for ease of application, which may reduce variability and increase recovery in the immunoprecipitation procedure. Additionally, protocols for the cross-linking of antibodies to DynaBeads are available. 9. It is generally anticipated that ChIP-Seq will be performed with proteins that previously have been successfully ChIPed and analyzed by direct, quantitative (or semiquantitative) PCR analysis of known binding and nonbinding sites. The efficacy of the current experiment can be similarly tested before proceeding. We recommend diluting 1 μL of the IP DNA (from Subheading 3.4, step 7) with 39 μL of TE and using 1 μL of this dilution for each analytical PCR reaction. The exact PCR conditions used will be based on methods previously established in the individual laboratory. 10. When assessing the size distribution of libraries, it is important to consider that ligation of primer adapters during library preparation increases the size distribution of the library by the
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total length of the adapters (e.g., a total increase of ~80 bp in preparations for Illumina Hi-Seq). Moreover, primers used in the amplification step of library preparation can form dimers which can be observed in BioAnalyzer traces. Care must be taken to remove as much primer dimer as reasonably possible as they will be sequenced if present in the final library, thus reducing the total number of useful reads.
Acknowledgments We thank J. Dalton for the assistance in establishing lab sequencing protocols. The work was supported by NIH grants 5R01-GM065494 (to O.M.A.), P50-HG002790 (A.Z.O.), and P30CA014089 from the National Cancer Institute (to USC Norris Cancer Center) and by NSF-MRI award #0923513 (to S. Nuzhdin) and by a pilot grant from the USC Epigenome Center sponsored by the Whittier Foundation (to O.M.A.). References 1. Hecht A, Grunstein M (1999) Mapping DNA interaction sites of chromosomal proteins using immunoprecipitation and polymerase chain reaction. Methods Enzymol 304: 399–414 2. Aparicio OM (1999) In: Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (eds) Current protocols in molecular biology. Immunoprecipitation for determining the association of proteins with specific genomic sequences In Vivo John Wiley and Sons Inc, New York. pp 21.3. 1–21.3.12 3. Hayashi M, Katou Y, Itoh T, Tazumi A, Yamada Y, Takahashi T, Nakagawa T, Shirahige K, Masukata H (2007) Genome-wide localization of pre-RC sites and identification of replication origins in fission yeast. EMBO J 26: 1327–1339 4. Xu W, Aparicio JG, Aparicio OM, Tavare S (2006) Genome-wide mapping of ORC and Mcm2p binding sites on tiling arrays and identification of essential ARS consensus sequences in S. cerevisiae. BMC Genomics 7:276 5. Wyrick JJ, Aparicio JG, Chen T, Barnett JD, Jennings EG, Young RA, Bell SP, Aparicio OM (2001) Genome-wide distribution of ORC and MCM proteins in S. cerevisiae: high- resolution mapping of replication origins. Science 294:2357–2360 6. MacAlpine DM, Rodriguez HK, Bell SP (2004) Coordination of replication and
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