Chromatin Immunoprecipitation Assay in Primary Mouse Hepatocytes and Mouse Liver

Abstract

Non-alcoholic steatohepatitis (NASH) is an advanced form of non-alcoholic fatty liver disease characterized by hepatosteatosis, liver cell injury, and inflammation. The pathogenesis of NASH involves dysregulated transcription of genes involved in critical processes in the liver, including metabolic homeostasis and inflammation. Chromatin immunoprecipitation (ChIP) utilizes antibody-mediated immunoprecipitation followed by the detection of associated DNA fragments via real-time PCR or high-throughput sequencing to quantitatively profile the interactions of proteins of interest with functional chromatin elements. Here, we present a detailed protocol to study the interactions of DNA and chromatin-associated proteins (e.g., transcription factors, co-activators, co-repressors, and chromatin modifiers) and modified histones (e.g., acetylated and methylated) in isolated primary mouse hepatocytes and mouse liver. The application of these methods can enable the identification of molecular mechanisms that underpin dysregulated hepatic processes in NASH.

1. Introduction

Chromatin immunoprecipitation (ChIP) is a powerful method to study the interaction between chromatin-associated proteins and functional chromatin elements in vivo [1, 2]. In a ChIP assay, the interaction between chromatin-associated proteins and DNA is first stabilized by cross-linking, before the chromatin is fragmented by physical sonication (X-ChIP) [3] or the chromatin is fragmented by enzymatic treatment without cross-linking (N-ChIP) [4]. The DNA and protein of interest are then co-immunoprecipitated by an antibody specific to the protein of interest and the enriched DNA sequence is detected by a variety of downstream methods, including real-time PCR, microarray, and sequencing [1]. Therefore, the ChIP assay enables the identification of genomic binding sites of transcription factors, their co-activators or repressors, chromatin modifiers, and post-translationally modified histones (e.g., by acetylation, methylation, ubiquitination, and more). The quantification following ChIP enables the enrichment of these chromatin-associated proteins to the targeted binding sites under two or more experimental conditions to be compared, or in the case of next-generation sequencing, enables non-biased profiling of genome-wide binding sites.

The ChIP assay was first developed by Gilmour and colleagues in 1984 as a tool to assess the association between RNA polymerase II and transcriptionally active genes in E. coli and Drosophila [5–7]. Later, the assay was modified to study the interaction between transcription factors, histones, and modified histones, and was used in mammalian cells [8–11]. Currently, it is widely used to assess changes in the binding of chromatin-associated proteins to native chromosomes in vivo, which occurs during gene transcriptional regulation, under both physiologic and diseased states. It is a powerful tool to elucidate the molecular mechanisms underlying the development and etiology of NASH [12–14].

This chapter provides a detailed protocol for cross-linking ChIP. We cover procedures of cross-linking, sonication, immunoprecipitation, and detection of a target gene(s) of interest using real-time PCR in primary mouse hepatocytes and mouse liver. We also provide notes and troubleshooting tips for critical steps throughout the procedure.

2. Materials

All solutions should be prepared using ultrapure water (e.g., the resistivity of 18.2 MΩ cm water by Millipore), stored at room temperature (RT), or at 4 °C or −20 °C, as indicated.

2.1. Cross-Linking

1. Healthy primary mouse hepatocytes in 15 cm dishes (1.5 × 10⁷ cells).

2. Frozen or freshly collected mouse liver tissues.

3. 1× Phosphate-buffered saline (PBS), autoclaved or sterilized via 0.22 μm filtration.

4. 37% formaldehyde stock, diluted in 1× PBS to 1% (v/v) prior to use.

5. 2 M glycine stock (to make a final concentration of 0.125 M).

6. Protease inhibitor and phosphatase inhibitor cocktails (e.g., Sigma protease inhibitor tablets and phosphatase inhibitor tablets). Make 100× stock in water and store at −20 °C prior to use.

7. 1 M Dithiothreitol (DTT) prepared as a 100× stock and stored at −20 °C.

8. DTT buffer: 100 mM Tris–HCl pH 9.4, 10 mM DTT. Add 10 mM DTT and 1× protease and phosphatase inhibitor cocktail immediately prior to use.

9. Cell lysis buffer I: 10 mM HEPES pH 6.5, 10 mM ethylenediaminetetraacetic acid (EDTA), 0.5 mM ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 0.25% Triton X-100. Store at 4 °C. Add 1× protease and phosphatase inhibitor cocktail immediately prior to use.

10. Cell lysis buffer II: 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 mM HEPES, pH 6.5. Store at 4 °C. Add 1× protease and phosphatase inhibitor cocktail immediately prior to use.

11. Hypotonic buffer: 10 mM HEPES pH 7.9, 1.5 mM magnesium chloride (MgCl₂), 10 mM potassium chloride (KCl), 0.2% IGEPAL CA-630, 1 mM EDTA pH 8.0, 5% sucrose. Store at 4 °C. Add 1× protease and phosphatase inhibitor cocktail immediately prior to use.

12. Cushion Buffer: 10 mM Tris–HCl pH 7.5, 15 mM sodium chloride (NaCl), 1 mM EDTA pH 8.0, 60 mM KCl, 10% sucrose. Store at 4 °C. Add 1× protease and phosphatase inhibitor cocktail immediately prior to use.

13. 1.5 mL Eppendorf tubes (sterile, DNAse-free and RNAse-free).

14. 15 mL and 50 mL conical tubes (sterile, DNAse-free and RNAse-free).

15. Weighing boats.

16. Razor blades.

2.2. Sonication

1. 20% sodium dodecyl sulfate (SDS) stock.

2. Sonication buffer: 50 mM Tris–HCl pH 8.0, 2 mM EDTA pH 8.0. Store at 4 °C. Add SDS to a final concentration of 1% and 1× protease and phosphatase inhibitor cocktail immediately prior to use.

3. 1.5 mL Eppendorf tubes (sterile, DNAse- and RNAse-free).

4. Cold ice bath.

2.3. Immunoprecipitation

1. Dilution buffer: 20 mM Tris–HCl pH 8.0, 2 mM EDTA pH 8.0, 167 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate. Store at 4 °C and add 1× protease and phosphatase inhibitor cocktail immediately prior to use.

2. RIPA buffer: 20 mM Tris–HCl pH 8.0, 2 mM EDTA pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate. Store at 4 °C.

3. 1% BSA in PBS, sterilized via 0.22 μm filtration. This will be used to prepare agarose beads for IP.

4. 10 mg/mL sheared salmon sperm DNA (Thermo Fisher Scientific). This will be used to prepare agarose beads for IP.

5. Protein A/G agarose beads (Santa Cruz Biotechnology protein A/G plus agarose) pre-blocked with BSA and sheared DNA.

6. Antibodies of interest (targeting a transcriptional factor, co-regulator, chromatin modifier, or modified histone), as well as an IgG negative control produced in the same species as the experimental antibody.

7. TSE I wash buffer: 1% Triton X-100, 2 mM EDTA pH 8.0, 20 mM Tris–HCl pH 8.0, 150 mM NaCl. Store at 4 °C and add up to 0.1% SDS before use.

8. TSE II wash buffer: 1% Triton X-100, 2 mM EDTA pH 8.0, 20 mM Tris–HCl pH 8.0, 500 mM NaCl. Store at 4 °C and add up to 0.1% SDS before use.

9. TSE III wash buffer: 250 mM LiCl, 1% IGEPAL CA-630, 1% sodium deoxycholate, 1 mM EDTA pH 8.0, 10 mM Tris–HCl pH 8.0. Store at 4 °C.

10. TE buffer: 1 mM EDTA, 10 mM Tris–HCl pH 8.0.

11. 1.5 mL Eppendorf tubes (sterile, DNAse- and RNAse-free).

2.4. Elution and Reverse Cross-Linking

1. Elution buffer: 1% SDS, 0.1 M sodium bicarbonate (NaHCO₃) prepared immediately prior to use at RT.

2. 5 M NaCl stock (to make a final concentration of 200 mM).

3. 1 M Tris–HCl pH 6.5 stock.

4. 20 mg/mL RNAase A.

5. 20 mg/mL proteinase K.

6. 1× TAE buffer: 40 mM Tris, 20 mM acetic acid, 1 mM EDTA.

7. 1% agarose gel made with 1× TAE containing 1× DNA stain (Biotium Gel Red DNA stain or equivalent).

8. 6× DNA loading buffer: 30% (v/v) glycerol, 0.25% (w/v) bromophenol blue, 0.25% (w/v) xylene cyanol FF.

9. DNA ladder (New England Biolabs 1 kb plus ladder or equivalent).

10. 1.5 mL Eppendorf tubes (sterile, DNAse- and RNAse-free).

2.5. Purification of DNA Fragments and qPCR

1. DNA purification kit (Qiagen QIAquick PCR purification or equivalent).

2. Validated primers for the promoters of interest and a negative control.

3. 2× SYBR Green PCR master mix (Thermo Fisher Scientific or equivalent).

4. 384 well real-time PCR plates.

2.6. Equipment

1. Liquid nitrogen-cooled mortar and pestle (Bel-Art).

2. Dounce homogenizer.

3. Shaker or rocker.

4. Refrigerated desktop centrifuges for 1.5, 15, or 50 mL tubes, or 384-well PCR plates.

5. Centrifuge for 1.5 mL tubes at RT.

6. Sonicator (Thermo Fisher Scientific Sonic Dismembrator or equivalent).

7. Rotator.

8. Water bath.

9. Spectrophotometer (NanoDrop 1000 or equivalent).

10. DNA gel electrophoresis system.

11. DNA gel imaging system (Kodak Gel Logic Imaging System or equivalent).

12. Quantitative real-time PCR instrument (Life Technologies QuantStudio 6 Flex PCR detection system or equivalent).

3. Methods

Perform cross-linking and elution at RT. Keep each solution needed for chromatin preparation and immunoprecipitation on ice and perform these procedures at 4 °C or on ice.

3.1. Cross-Linking in Primary Hepatocytes

1. Start with two 15 cm dishes of healthy primary mouse hepatocytes (1.5 × 10⁷ cells) per treatment (see Notes 1 and 2).

2. Wash cells with 5 mL of RT PBS twice.

3. Add 10 mL of 1% formaldehyde in PBS (mix 0.27 mL of 37% formaldehyde stock with 10 mL of RT PBS in a chemical hood) per dish and incubate the cells on a shaker/rocker for 10 min at RT (see Note 3).

4. Stop the reaction by adding 625 μL of 2 M glycine stock to a final concentration of 125 mM and incubate the cells on a shaker/rocker for 5 min at RT (see Note 4).

5. Rinse cells with 5 mL of cold PBS twice.

6. Scrape cells into 5 mL of cold PBS and collect cells in 15 mL tubes. Centrifuge at 1000 × g for 5 min at 4 °C.

7. Carefully aspirate the supernatant and suspend the cell pellet in 300 μL of cold DTT buffer. Vortex cells for 10 s and incubate the cells on ice for 10 min, mix by vertexing every 2–3 min. Centrifuge at 2000 × g for 3 min at 4 °C.

8. Suspend the pellet into 300 μL of cold cell lysis buffer I. Vortex and incubate on ice for 10 min, mix by inverting every 2–3 min. Centrifuge at 2000 × g for 3 min at 4 °C.

9. Suspend the pellet into 300 μL of cold cell lysis buffer II. Vortex and incubate on ice for 10 min, mix by inverting every 2–3 min. Centrifuge at 2000 × g for 3 min at 4 °C.

10. Suspend the pellet into 450 μL of sonication buffer containing the protease and phosphatase inhibitor cocktail, sonicate, and proceed to Subheading 3.3.

3.2. Cross-Linking in Mouse Liver Tissues

1. Start with 100–200 mg frozen or fresh mouse liver tissues per treatment. For frozen livers, grind the tissue into small chunks (1–3 mm²) in liquid nitrogen using a cooled mortar and pestle and collect into 50 mL tubes. Keep all tubes on dry ice until all samples are ready to proceed to cross-linking. For fresh liver tissues, cut the tissues into small chunks using a razor blade on a weighing boat on ice and proceed to cross-linking in 50 mL tubes (see Note 5).

2. Move tubes to RT and add 10 mL of 1% formaldehyde in PBS (mix 270 μL of 37% formaldehyde stock with 10 mL of PBS at RT) per tube and incubate on a rocker for 15 min at RT (see Note 3).

3. Stop the reaction by adding 625 μL of 2 M glycine stock to a final concentration of 125 mM and incubate the tissue on a shaker/rocker for 5 min at RT (see Note 4).

4. Centrifuge at 1000 × g for 5 min at 4 °C. Wash the pellet twice with 10 mL of ice-cold PBS. Centrifuge for 5 min at 1000 × g at 4 °C and discard wash buffer.

5. Add 550 μL of cold hypotonic buffer containing the protease and phosphatase inhibitor cocktail. Grind the tissue in a Dounce homogenizer (15–25 strokes) on ice to release nuclei (see Note 6).

6. Add 550 μL of cold cushion buffer containing the protease and phosphatase inhibitor cocktail to a 1.5 mL tube and lay the homogenates from step 5 on the top of the cushion buffer. Do not mix the two layers. Centrifuge at 500 × g for 1 min at 4 °C to pellet the nuclei.

7. Suspend the pellet into 900 μL of sonication buffer containing the protease and phosphatase inhibitor, divide into two 1.5 mL tubes, and proceed to Subheading 3.3.

3.3. Sonication

1. Sonicate the cells in 1.5 mL tubes with 15 s on/1 min off pulses at 30% output for 6–20 cycles on an ice bath (see Note 7). The desired chromatin length is between 200 and 1000 bp, see Fig. 1. Sonication conditions and cycles must be determined empirically (see Note 8).

2. Centrifuge at 10,000 × g for 1 min at 4 °C, and carefully combine and transfer the supernatant from sonicated chromatin to a new tube while avoiding cell debris. Store obtained chromatin at −80 °C for up to 2 months or proceed to the next steps.

3. Remove 50 μL of each sonicated sample as Input. It will be used to determine chromatin size and quantify DNA concentration as described in Subheading 3.4 and to calculate relative enrichment in the real-time PCR step as described in Subheading 3.7 (see Note 9).

Fig. 1.

Chromatin lengths after sonication. Sonicated and purified chromatin from mouse liver was run on a 1% agarose-TAE gel. Sheared DNA fragments are between 0.2 kb and 1 kb in length. Lane 1 DNA; lane 2 DNA ladder

3.4. Reverse Cross-Linking and Determination of Chromatin Size and Concentration

1. Add 2 μL of RNAse A (final concentration of 0.4 mg/mL) to input samples from step 3 in Subheading 3.3 (see Note 10).

2. Add 5 M NaCl to a final concentration of 200 mM, 1 M Tris–HCl pH 6.5 to a final concentration of 10 mM, and 2.5 μL of 20 mg/mL proteinase K to each tube and mix (see Note 11).

3. Incubate samples in a 65 °C water bath for 4–6 h or overnight to reverse cross-link and to digest protein (see Note 12).

4. Purify DNA using a DNA purification kit following manufacturer’s instructions. Elute DNA into 75 μL of water (see Note 13).

5. Measure DNA concentration using a spectrophotometer.

6. Mix 5 μL of purified DNA with DNA loading buffer and run samples on a 1% agarose-TAE gel with Gel Red DNA stain, along with a DNA ladder. Determine chromatin size (see Note 8), see Fig. 1.

3.5. Chromatin Preclearing and Immunoprecipitation

1. Dilute the sonicated chromatin from step 2 in Subheading 3.3 to a final 10× volume with the dilution buffer containing 1× protease and phosphatase inhibitor cocktail (e.g., 450 μL of sonicated chromatin + 4050 μL of dilution buffer) (see Note 14).

2. Prepare protein A/G beads pre-adsorbed with BSA and sheared salmon sperm DNA according to steps 3–5.

3. Wash commercial protein A/G beads twice with RIPA buffer.

4. Centrifuge at 2000 × g for 1 min and remove the supernatant. Add sheared salmon sperm DNA to a final concentration of 75 ng/μL and BSA to a final concentration of 0.1 μg/μL beads.

5. Add RIPA buffer to 2× the bead volume and incubate on a rotator at RT for 30 min (see Note 15).

6. To pre-clear the chromatin (see Note 16), add 3–5 μg IgG and 40–80 μL beads (see Note 17) to the diluted chromatin from step 1 for 1 h at 4 °C with rotation. Centrifuge chromatin samples at 2000 × g for 1 min at 4 °C. Transfer the supernatant to a fresh tube.

7. Start with 50–150 μg chromatin per IP in 1.5 mL tubes. Add the primary antibody or IgG-negative control to each IP. Typically, 1–5 μg antibody per 50 μg pre-cleared chromatin (see Notes 18 and 19) is sufficient and should be determined empirically. At least three IP/antibody replicates are recommended allowing the determination of statistical significance (see Note 20).

8. Incubate on a rotator at 4 °C overnight.

3.6. Washing, Eluting, and Reverse Cross-Linking

1. Collect the antibody-protein-DNA complex by adding 25 μL/IP protein A/G beads pre-soaked with DNA and BSA (steps 3–5, Subheading 3.5), at 4 °C for 2 h with rotation (see Note 17). Centrifuge at 2000 × g for 1 min at 4 °C and discard the supernatant.

2. Wash the beads sequentially with 500–1000 μL each of TSE I, TSE II, and TSE III buffer, and twice with 500 μL of TE buffer, 5–10 min each at 4 °C (see Note 21).

3. Centrifuge at 2000 × g for 1 min at 4 °C and carefully discard the supernatant.

4. Add 125 μL of freshly prepared elution buffer to the beads, briefly vortex, and incubate at RT for 30–60 min with rotation. Ensure that the tubes are tightly closed.

5. Centrifuge at 2000 × g for 1 min at RT and collect the supernatant.

6. Perform reverse cross-linking and DNA purification as described in steps 1–4, Subheading 3.4 (see Notes 10–13).

3.7. PCR Quantification

1. Use purified IP DNA samples from step 6 in Subheading 3.6 (75 μL) and Input from step 4 in Subheading 3.4 (dilute twofold with H₂O to 150 μL) for PCR.

2. Perform real-time PCR using primers for the promoter regions of interest and positive and negative control (e.g., a house keeping gene promoter region) using a standard PCR protocol (see Note 22). The reactions can be run using 1–2 μL of samples with 2× SYBR Green master mix in triplicate for each pair of primers (final concentration of 250 nM each primer) in a 384-well plate with default settings for 45 cycles (see Note 23).

3. Normalize the relative signal (threshold CT values from triplicates) of the ChIP samples to the signaling from the respective Input samples using 2^{(Input CT – ChIP CT)}.

4. Calculate the relative enrichment of a protein of interest on a specific promoter by normalizing the expression to the control IgG, or in the case of multiple treatments/groups, to the control IgG of the control group. Appropriate statistical analyses can be applied to determine whether the enrichment of the protein of interest is significantly higher relative to IgG, and whether the enrichment differs among treatments/groups (see Notes 24 and 25).

4. Notes

1. It is important to use an established, two-step collagenase perfusion protocol to obtain high-quality primary mouse hepatocytes for ChIP [15]. A typical monolayer hepatocyte culture should be harvested for analyses within 48 hours of isolation, as hepatocytes lose their morphology, hepatocyte-specific gene expression, and responses to hormones and pharmacologic treatments over time.

2. We recommend including a positive treatment/control in the ChIP assay, where the enrichment of the protein of interest is altered.

3. Standard cross-linking time is 10–15 min. However, the optimal cross-linking time should be empirically determined. Although sufficient cross-linking to stabilize DNA–protein interactions is necessary for ChIP to work, excess cross-linking can result in reduced antigen availability for the antibody, increased non-specific interactions, and can affect downstream steps including nuclei isolation, sonication, and DNA recovery.

4. The addition of glycine is a necessary step to quench the cross-linking reaction by formaldehyde.

5. It is important that the hepatocytes and liver tissue samples do not reach high temperatures to prevent sample degradation by proteases. When collecting mouse liver tissue, the tissue should be snap-frozen in liquid nitrogen and stored at −80 °C until use. Avoid repeated freezing and thawing of the tissue.

6. The number of strokes during homogenization of liver tissues in hypotonic buffer needs to be empirically determined as this step breaks the cell membrane open to isolate intact nuclei. Use a microscope to determine optimal homogenization that lyses cells but not nuclei.

7. Sonication generates heat. Therefore, it is critical that samples are kept in a cold ice bath (or a salt/ice bath) during sonication and are allowed enough time to cool down between each sonication.

8. Obtaining an optimal range of chromatin lengths is critical (see Fig. 1 as an example). DNA fragments longer than 1.5 kb tend to increase background due to their non-specific binding to antibodies. DNA fragments less than 100 bp will reduce signaling as they may not be amplified by PCR. The volume of samples, depth of the probe, sonication duration, and sonication strength all affect sonication results. Therefore, we recommend keeping consistency in volume, depth of the probe, and sonication strength, and altering sonication times while optimizing. It is essential to obtain an optimized sonication condition before performing IP.

9. It is important to have input samples. They will be used to determine chromatin size and to calculate relative enrichment in real-time PCR. If multiple treatment groups are studied, the size and concentration of sheared chromatin in each Input, as well as the abundance of DNA fragments of interest quantified by real-time PCR in each input, should be similar. Optimization would be needed to match these to avoid artifacts and to obtain reproducible results.

10. RNA should be removed by RNAse A as high levels of RNA will interfere with DNA purification using a DNA purification kit.

11. The optimal condition for proteinase K is under denaturation (e.g., in the presence of SDS), pH 6.5–9.5, and in the presence of EDTA.

12. Cross-linking by formaldehyde is reversed by heat. Successful reverse cross-linking is necessary for downstream DNA recovery and quantification by real-time PCR.

13. Other commonly used purification methods include the extraction of DNA using phenol:chloroform:isoamyl alcohol (25: 24:1, v/v/v) followed by ethanol precipitation.

14. Sonication buffer contains high SDS and no salt. The addition of the dilution buffer makes the final SDS and salt concentration suitable for IP.

15. Protein A or G beads should be used for maximal binding of the beads to the antibody of interest as they have different affinities to antibodies produced in different species. Therefore, we recommend mixing equal amounts of protein A and G beads, and incubating with BSA and sheared salmon sperm DNA. It is important to pre-adsorb protein A/G beads with BSA and sheared DNA to reduce non-specific interaction. Magnetic beads can also be used.

16. The pre-clearing step using BSA and sheared DNA pre-adsorbed beads removes the proteins and DNA that non-specifically bind to the beads, and therefore reduces background and increases signal-to-noise ratio.

17. Mix the bead suspension well before adding to samples. Cut the ends of pipette tips to make the orifice larger to transfer beads.

18. The relative sensitivity of antigen epitopes to formaldehyde cross-linking should be taken into consideration. Antibodies that work well for IP of a native protein may not work for ChIP. Therefore, we recommend using ChIP-grade antibodies tested by vendors or peers. It is important to validate the antibody and include control samples that lack the protein of interest (e.g., knockout or knockdown) or a known treatment that will increase or decrease the enrichment of the targeted protein to a known gene promoter. In addition, the amount of each antibody used for IP should be empirically determined.

19. It is important to include IgG produced in the same species as the antibody targeting the protein of interest as a negative control. The IgG should pull down a negligible amount of DNA fragments of interest.

20. We recommend performing three to six technical repeats of each antibody.

21. Efficient washing is critical to reduce background. The outcome of the wash depends on the concentrations of the salt and detergent (particularly SDS), the volume of the washing buffer, and the duration of the wash. If high background is observed after real-time PCR quantification (low CT in control IgG-treated samples), a higher amount of SDS in TSE I and II buffer, a higher volume of washing buffer, and additional washes may be needed. If low or no signaling is observed, reduce SDS, wash time, and volume.

22. Real-time PCR primers targeting a gene(s) of interest and a control housekeeping gene (or a region of the target gene that does not bind the protein of interest) can be designed using online tools (e.g., Primer 3) or manually. We recommend designing primers of 18–22 nucleotides in length and with 35–55% GC nucleotides. Determine primer efficiency using serial dilutions of the Input and including melting curve analysis in the PCR program. A suitable primer pair should show an amplification efficiency ~90–110%, a single sharp melting peak, and amplification of a DNA fragment 75–120 bp in size.

23. A final concentration of ~250 nM of each primer is typically used. However, titration of primer concentrations between 100 and 500 nM may be required to obtain optimal results.

24. We recommend including three to six technical repeats of IP per antibody in each ChIP assay and performing at least two to three biological repeats of ChIP assays before reaching a conclusion.

25. The ChIP samples can also be used for other analyses, including library preparation for a ChIP-sequencing analysis, which reveals genome-wide binding sites.