Chromatin preparation from tissues for chromatin immunoprecipitation (ChIP)

This chromatin preparation from tissues protocol provides a validated and reproducible approach for extracting high-quality chromatin from a range of tissue samples, suitable for downstream chromatin immunoprecipitation (ChIP) applications. The workflow begins with tissue preparation as the initial step, followed by optimized cross-linking, tissue dissociation, cell lysis, and chromatin shearing, tailored for challenging sample types like liver, brain, adipose tissue with high lipid content, and other complex tissues.

Introduction to chromatin immunoprecipitation

Chromatin immunoprecipitation (ChIP) is a central technique for studying epigenetic regulation and protein-DNA interactions within biological tissues. Reliable chromatin preparation from tissues is essential for successful ChIP experiments, enabling scientists to investigate histone modifications, transcription factor occupancy, and chromatin accessibility using foundational molecular biology techniques. The chromatin preparation from tissues protocol was developed to overcome challenges specific to tissue samples—including variable cell density, endogenous nuclease activity, and abundant background proteins—by providing a straightforward workflow from initial tissue handling to finely sheared chromatin ready for immunoprecipitation. Designed for compatibility with both cross-linked and native ChIP workflows, this protocol is designed to minimize sample loss, reduce background, and preserve critical protein-DNA interactions, which are crucial for capturing protein-protein interactions in chromatin studies. Protein cross-linking and chromatin binding are essential for stabilizing these complexes during ChIP.

Background and principles

Effective chromatin preparation from tissue lays the groundwork for accurate and reproducible ChIP assays. The core principle involves stabilizing native protein-DNA complexes—usually via formaldehyde cross-linking—to capture a snapshot of biological interactions. Formaldehyde primarily reacts with lysine residues during  in vivo  crosslinking, enabling the efficient capture of protein-DNA and protein-protein interactions. Following cross-linking, tissue dissociation, and cell lysis, chromatin is released; degradation is minimized by incorporating protease inhibitors throughout the workflow. Mechanical and enzymatic shearing (using sonication or micrococcal nuclease) produces optimal chromatin fragment sizes, which are critical for resolution in downstream ChIP-qPCR and ChIP-seq studies. The yield and quality of chromatin are critical for downstream ChIP applications. Our recommended approach standardizes variables such as tissue mass, buffer composition, and shearing conditions, thereby establishing a platform for reliable comparison across different tissue types. Maintaining low temperatures and rapid processing helps preserve the integrity of chromatin.

It is recommended that 30 mg of liver tissue be used for each ChIP/antibody. However, this amount may vary for other tissues. The exact amount of tissue depends upon protein abundance, antibody affinity, and cross-linking efficiency.

This protocol was optimized using 5-15 µg chromatin for each ChIP assay. The exact chromatin concentration should be determined for each tissue type before starting the X-ChIP assay. It is also crucial to quantify the input DNA and include an input sample as a control in ChIP-seq workflows to ensure accurate normalization and peak validation. Our cross-linking chromatin immunoprecipitation (X-ChIP) protocol should be used after the chromatin preparation detailed below. Protease inhibitors should be included in all solutions, including PBS PMSF 10 µL/mL, aprotinin 1 µL/mL, and leupeptin 1 µL/mL.

Adapted from protocols kindly provided by Henriette O’Geen, Luis G. Acevedo and Peggy J. Farnham.

Materials:

• FA lysis buffer
• 50 mM HEPES-KOH pH 7.5
• 140 mM NaCl
• 1 mM EDTA pH 8
• 1% Triton X-100 or 1% NP-40
• 0.1% Sodium deoxycholate
• 0.1% SDS
• Protease inhibitors (add fresh each time)

Stage 1 - Cross-linking

Steps

Thaw frozen tissues on ice.

• This process can take hours, depending on the amount of tissue.
• Tissue should be cut in a petri dish resting on a block of dry ice.

Chop frozen or fresh tissue into small pieces using two razor blades.

• The piece should measure between 1-3 mm³.

Transfer tissue into a 15 mL conical tube.

• Weigh the empty tube before and after tissue transfer to calculate the amount of tissue available.

Prepare cross-linking solution in a fume hood.

• Use 10 mL PBS per gram of tissue.
• Add formaldehyde to a final concentration of 1.5 % and rotate the tube at room temperature for 15 min. The Formaldehyde percentage and length of time for cross-linking might need to be optimized for different tissues.

Stop the cross-linking reaction by adding glycine to a final concentration of 0.125 M.

• Continue to rotate at room temperature for 5 min.

Centrifuge tissue samples for 5 min at 100 x g at 4°C.

Aspirate media and wash with 10 mL ice-cold PBS.

• Centrifuge for 5 min at 100 x g at 4°C and discard the wash buffer.

Stage 2 - Tissue disassociation

Steps

The Medimachine from Becton Dickinson can be used to obtain a single cell suspension.

• Use 2 medicons (50 μm) per gram of tissue to process.

Cut a 1 ml pipette tip to make the orifice larger.

Add between 50-100 mg (3-4 chunks) of tissue resuspended in 1 mL of PBS.

Add this solution to the medicon and grind tissue for 2 min.

Collect cells from the medicon by inserting an 18-gauge blunt needle and a 1 mL syringe.

• • Collect cells in a conical tube on ice.

Repeat from step two until all the tissue is processed.

Check the cell suspension using a microscope to ensure a unicellular suspension is obtained.

Centrifuge cells for 10 min at 300 x g at 4 °C.

Measure/estimate cell pellet volume for the next step.

Carefully aspirate off supernatant and resuspend pellet in FA lysis buffer.

• We recommend 750 μl of lysis buffer per 1x10⁷ cells.

Alternatively a Dounce homogenizer (pestle A) can be used for some soft tissues.

Add 50-100 mg of tissue resuspended in 1 mL PBS with protease inhibitors and disrupt the tissue with a Dounce homogenizer until the tissue is processed (typically >30 passes).

Tissue selection and preparation

Selecting and preparing the right tissue samples is a foundational step for successful chromatin immunoprecipitation (ChIP) experiments. The integrity of chromatin structure and preservation of protein-DNA interactions depend heavily on how the tissue is handled prior to chromatin extraction. Researchers can use fresh, frozen, or formalin-fixed paraffin-embedded (FFPE) samples, each with unique considerations for ChIP assay performance.

• Fresh tissue: Fresh tissue is optimal for chromatin immunoprecipitation chip workflows, as it allows immediate fixation, preserving native protein-DNA complexes and minimizing degradation. Rapid processing ensures high-quality chromatin extraction and reliable detection of protein-DNA interactions.
• Frozen tissue: When fresh tissue is unavailable, frozen tissue is a robust alternative. Tissue should be snap-frozen in liquid nitrogen immediately after collection to lock in chromatin structure and maintain protein-DNA binding. Prior to ChIP, frozen tissue is carefully thawed and processed, following protocols that mirror those for fresh tissue to ensure consistent chromatin yield.
• FFPE tissue: Due to their long-term stability, FFPE samples are widely used in clinical and archival research. However, the fixation process can complicate chromatin extraction, often resulting in lower yields and more challenging recovery of protein-DNA complexes. Specialized protocols, such as Chrom-EX PE, have been developed to enhance chromatin immunoprecipitation from FFPE tissue samples. This protocol should not be used for FFPE tissue.

Careful tissue selection and preparation are essential for maximizing the success of ChIP assays, whether investigating gene regulation in normal and cancer cells, profiling histone modifications, or mapping transcription factor binding across genomic regions.

Safety precautions

Safety is paramount when performing chromatin immunoprecipitation, as the protocol involves hazardous chemicals and potentially biohazardous tissue samples. Adhering to the following precautions ensures a safe laboratory environment:

• Handling of formaldehyde: Formaldehyde is toxic and volatile; always handle it in a certified fume hood while wearing appropriate personal protective equipment (PPE), including gloves, lab coat, and safety goggles. Dispose of formaldehyde waste according to institutional and local regulations.
• Biohazardous materials: Tissue samples, especially those derived from human or animal sources, may contain infectious agents. Always use gloves, lab coats, and eye protection, and follow biosafety guidelines for handling and disposal of biological materials.
• Chemical disposal: Dispose of all chemicals, including lysis buffers, cross-linking reagents, and organic solvents, in accordance with local environmental and safety regulations. Never pour hazardous chemicals down the drain.

By following these safety guidelines, researchers can minimize risks and ensure the safe execution of chromatin immunoprecipitation protocols.

Comparison to other methods

Chromatin preparation from tissues, as outlined in this protocol, employs optimized cross-linking and shearing strategies that set it apart from traditional cell-based methods. Unlike protocols for cultured cells, tissue-based preparations must account for heterogeneous structure, variable protein levels, and the need for effective tissue dissociation techniques. Mechanical dissociation (eg, using a Medimachine or dounce homogenizer) is often necessary prior to lysis and shearing. Compared to alternative fragmentation methods, such as enzymatic MNase digestion, mechanical sonication offers better reproducibility for complex or fibrous tissue. While MNase provides nucleosome-level resolution, it can introduce sequence bias and be sensitive to tissue-specific chromatin accessibility. This protocol integrates best practices from both approaches, balancing the efficiency of fragmentation and the preservation of protein-DNA complexes, while minimizing background and enabling high signal-to-noise immunoprecipitation.

Applications

High-quality chromatin prepared from tissues is indispensable for advanced epigenetic research, including both targeted and genome-wide studies. The chromatin preparation from tissues protocol is widely utilized in applications such as ChIP-qPCR, ChIP-seq, and histone modification mapping, enabling detailed analysis of transcription factor binding, chromatin remodeling, and the identification of regulatory elements across various tissue types. Its use extends to studies investigating developmental biology, disease epigenetics (such as cancer, metabolic, and neurological disorders), and tissue-specific gene regulation. Reliable preparation of chromatin from frozen archives also supports retrospective studies on clinical samples. By providing consistent yields and chromatin fragment sizes, this protocol ensures compatibility with next-generation sequencing platforms and enrichment analyses. Researchers benefit from streamlined experimental workflows, reproducible ChIP results, and the ability to interrogate subtle regulatory events in complex tissue environments.

Limitations

While this protocol enables the isolation of chromatin from tissues, users should be aware of the key limitations inherent to tissue-based ChIP experiments. The success of chromatin extraction can vary based on tissue type, sample freshness, and cross-linking efficiency, occasionally necessitating protocol optimization for specific tissues. High background levels may result from incomplete cell lysis or tissue heterogeneity, particularly in samples with fibrous or fatty tissue. Dependence on antibody specificity and quality remains a common bottleneck, and over- or under-fixation can compromise both chromatin yield and immunoprecipitation sensitivity. DNA shearing requires careful optimization to prevent the loss of high-molecular-weight fragments or epitope masking. Additionally, input requirements for tissue-based protocols are often higher than for cell culture, potentially limiting studies using very rare or archived material. Finally, some downstream applications require base-pair resolution or single-cell sensitivity that is unmatched by standard protocols, highlighting the need for ongoing technical refinement.

Troubleshooting

For optimal results with chromatin preparation from tissue samples, carefully address common troubleshooting points. Use high-quality, fresh or properly stored frozen tissue, and ensure effective, rapid cross-linking to minimize protein and DNA degradation. Consistently keep samples cold during all steps, especially during dissociation and lysis, to preserve chromatin integrity. If the chromatin yield is low, review the tissue mass, lysis efficiency, and freshness of the protease inhibitor. Troubleshoot excessive background or low ChIP enrichment by confirming complete cellular dissociation and sufficient chromatin shearing within the ideal fragment size range (200–700 bp). For poor antibody performance, validate antibody specificity in control ChIP assays and adjust concentrations as needed. Use negative and positive ChIP controls to benchmark assay efficiency. When encountering variable results, optimize sonication or MNase conditions, and analyze fragment distribution by electrophoresis.