Chromatin Immunoprecipitation ChIP

History of ChIP

Using antibodies to precipitate target proteins has its origins in the 1960s, when Barrett et al. referred to immunoprecipitation as a tool for measuring the concentration of gamma globulins in human serum, offering more precision than other techniques available at the time such as electrophoresis.^1,2 However, ChIP did not emerge until the 1980s when it was implemented by Gilmour & Lis as a technical proof of principle, using RNA polymerase and the lac operon in bacteria, crosslinked with UV radiation.³ This was followed by the same group with investigations into the activity of RNA polymerase II and topoisomerase I at heat shock genes in untreated or heat-shocked Drosophila melanogaster cells.^4–6 The use of UV in ChIP was later replaced by formaldehyde, a more efficient and reversible method of crosslinking protein to DNA.⁷ One of the major uses of ChIP today is to study the link between histone modifications and transcriptional activity. ChIP was first applied to this question in 1988, when Hebbes et al. found that acetylated histone H4 was enriched at genomic regions that were being actively transcribed, but not at a control locus.⁸

ChIP analysis initially relied on traditional molecular biology approaches such as Southern blots, later superseded by end-point PCR and qPCR.⁹ These techniques are useful if known genes or sequences are the subject of study, for instance to study how the association between a transcription factor and its binding sites changes with experimental treatment, but cannot reveal previously unknown regions of interaction. This limitation was overcome by ChIP-chip, in which ChIP was combined with microarray technology to provide a global view on protein-DNA interactions, first in yeast before being applied to mammalian cells in 2001.^10,11 Next-generation sequencing (NGS) combined with ChIP (ChIP-seq), first performed in 2007, has since become the most popular technique for analyzing ChIP experiments, providing a rapid, high-resolution snapshot of protein-DNA interactions.¹²

Chromatin, Epigenetics, and Targets of ChIP

The spatial organization of genomic material is essential for appropriate gene expression.¹³ In eukaryotes, DNA in the nucleus is complexed with histone proteins to form chromatin, which is essential for preventing DNA strands from becoming tangled and to enable DNA packaging. The primary unit of chromatin is the nucleosome, which consists of a core of 8 histone proteins (2 copies each of H2A, H2B, H3 and H4) around which 147 bp of DNA wraps approximately 1.75 times (Figure 1). The histone H1 protein is typically referred to as a linker and has numerous functions in chromatin including the stabilization of DNA wrapping around the nucleosome and assembly of higher order chromatin structures.¹⁴

Figure 1: Nucleosome structure and chromatin organization.The histone octamer is at the core of nucleosome structure, comprising an H3-H4 tetramer and two H2A-H2B dimers around which DNA wraps.¹⁵ Each histone protein has an unstructured N-terminal (as well as an unstructured C-terminal for H2A) that protrudes from the nucleosome core and is called a tail. Histone tails are accessible to histone modifying proteins and are the prime site for PTMs, which influence whether chromatin is structured as euchromatin (transcriptionally active) or heterochromatin (transcriptionally repressed). Acetylation is generally a marker for euchromatin, because it is negatively charged and so weakens the strength of attraction between the positively charged histones and DNA.¹⁶ Methylation has no effect on charge and can be associated with either transcriptional activation or repression depending on the site.¹⁶ For example, H3K4 methylation results in gene activation, whereas H3K27 methylation leads to gene silencing. Though PTMs of H3 and H4 are the best characterized, H2A and H2B PTMs also affect chromatin structure, including methylation and ubiquitination.¹⁷

Chromatin exists in distinct, spatially segregated states that influence how accessible it is to transcriptional machinery. Heterochromatin is a tightly packed form of chromatin that is essential for genomic stability and associated with repressed transcription.¹⁸ In contrast, euchromatin is less compact and more permissive of gene expression.¹⁹

Post-translational modifications (PTMs) of histones are essential in determining chromatin state. These PTMs include phosphorylation, methylation, acetylation, ubiquitination, sumoylation, ADP ribosylation and more, which are conferred upon the histones by specific enzymes termed readers, writers and erasers.²⁰ The combination of PTMs, particularly on histone lysine residues, are strongly associated with the dynamic regulation of gene expression by altering chromatin structure and recruiting remodeling enzymes.²¹

Histone PTMs are often referred to as epigenetic marks, heritable alterations to chromatin that regulate gene expression but do not change the underlying base sequence (though the exact definition has been debated).¹⁸ Other epigenetic marks include DNA methylation, chromatin remodeling, and non-coding RNA signaling.²² Unlike the base genome, epigenetic marks are dynamic, adapting to environmental cues and, along with transcription factor activity, control gene expression to determine cell fate and function.

ChIP has been used to understand the DNA binding properties of chromatin remodelers, histone-modifying enzymes, transcription factors and more. A successful ChIP experiment should take into account the nature of the ChIP target to determine the optimum approach. These include expression properties, such as how abundant the target protein is, the conditions under which it is expressed, and whether it is localized to the nucleus, and DNA-binding properties, such as if it binds to DNA directly or via another protein, how strong the association is, and how many binding loci are under investigation.

IP Formats

ChIP is one of a number of variations on the original IP format, which precipitates only a single protein target. Other IP formats enable the study of protein-protein (Co-IP), protein-DNA (ChIP) and protein-RNA (RIP) interactions. An overview of each method can be found in our IP Guide, while a comparison of the main features of each is below in Table 1.

Table 1:Key applications and differences between IP formats.

ChIP Workflow

ChIP differs from IP and co-IP because it aims to precipitate not only proteins, but also DNA, on the basis that proteins will bind to specific regions of DNA to regulate their expression or structure. The workflow of ChIP (Figure 2) is therefore distinct from that of IP and co-IP because it begins by cross-linking proteins to DNA, usually by formaldehyde treatment, preserving the state of protein-DNA interactions (for example, after a certain treatment). Cells are then lysed and sonicated to shear DNA, resulting in small pieces of DNA attached to DNA-binding proteins. The proteins can then be captured as in regular IP, targeted with specific antibodies immobilized on beads. Following dissociation of the DNA from the protein, the DNA is analyzed by PCR, microarray or sequencing.

Figure 2: Schematic of chromatin immunoprecipitation (ChIP) workflow.

ChIP offers advantages over other techniques for investigating protein-DNA interactions by virtue of reflecting the native, intracellular context of protein-bound DNA, as opposed to in vitro assays such as the electrophoretic mobility shift assay (EMSA), and can therefore account for the effects of epigenetics as well as known and unknown binding partners. Recently developed techniques that isolate protein-bound DNA under in vivo conditions include CUT&RUN and CUT&Tag,^23,24 which are advantageous because they have lower cell number input requirements than ChIP.

Native ChIP (N-ChIP)

The ChIP workflow described in Figure 2 (which is also called crosslinking ChIP or X-ChIP) can be performed in the absence of formaldehyde or any other crosslinking agent in a variant called Native ChIP (N-ChIP). Because proteins and DNA are not crosslinked, N-ChIP can typically only be used for very tight protein-DNA interactions, such as histone binding, but it will not result in false positives caused by crosslinking irrelevant proteins to DNA. While most of the N-ChIP workflow is the same as X-ChIP, native chromatin is fragmented using a nuclease, usually micrococcal nuclease (MNase), instead of sonication, which would be too harsh and disrupt histone-DNA binding.

N-ChIP has the advantage of improved antibody recognition because epitopes will not be masked by fixation. The immunoprecipitation step is often more efficient than in X-ChIP, meaning that PCR is not required for DNA analysis, more easily enabling quantification. However, N-ChIP can generally not be used for non-histone proteins due to their weaker interactions, and MNase digestion is not random like sonication, which may bias analyses.

Applications and Limitations of ChIP

ChIP is a powerful tool for understanding how proteins interact with chromatin in its native environment. Fundamentally, ChIP is useful for determining which DNA sequences are being bound by the target protein, such as a transcription factor. This could identify new transcription factor binding sites, suggesting an interaction with a gene previously not known to be regulated by that protein. From this, how DNA-protein interactions change with time, treatment, cellular activity and disease state can be investigated. ChIP can also be used for identification of genetic elements, either via transcription factor binding or inferring element function based on histone modifications at the site.²⁵

With regards to histone-DNA interactions, ChIP can identify higher order chromatin structures, such as sites of heterochromatin by trimethylation of H3 lysine 9 (H3K9me3), suggest activity or inactivity at genomic regions based on well-known histone modifications, and help determine the function of unexplored histone modifications.

Despite the discoveries enabled by ChIP, the technique has significant limitations. ChIP is a complex, multistep technique that has high cell input requirements and is slow to perform compared to comparable techniques such as CUT&RUN.²³ ChIP can yield low signal and have high background, which is particularly true if an antibody of insufficient specificity is used, such as one that cannot adequately distinguish between mono-, di- or tri-methylation of a histone. The resolution of ChIP is limited to the size of DNA fragment that is precipitated, typically ~150-500 bp, meaning precise binding sites cannot be determined without other techniques. Further, the function of a protein at a given site cannot be inferred from ChIP alone, such as if it is activating or repressing transcription. With X-ChIP, the protein may not be directly interacting with the DNA, the epitope of the protein could be masked by cofactors, while crosslinking can fix irrelevant proteins that only transiently interact with the DNA but have no functional significance.

Sample Preparation

Crosslinking Proteins to DNA

Crosslinking stabilizes and preserves protein-DNA interactions and is necessary when performing ChIP on any non-histone proteins. Histones can be precipitated without crosslinking in N-ChIP, although new modifications to the histones may occur in the absence of crosslinking if enzymes are still active.

Crosslinking in X-ChIP can be performed with UV light, formaldehyde, or other chemical crosslinkers, but formaldehyde is the standard approach because it is a reversible and highly efficient crosslinking agent. With a crosslinking distance of only 2 Å (0.2 nm), crosslinking by formaldehyde results in the preservation only of proteins closely associated with DNA, though it will also fix proteins to proteins, so cofactors can be co-precipitated to facilitate the study of indirect protein-DNA interactions. For more distal interactions or larger protein complexes, other crosslinkers such as DSG (7.7 Å) or EGS (16.1 Å) can be used instead.

Formaldehyde is typically used at 1% concentration at room temperature for 10 minutes, but a performing a time course pilot experiment is recommended using a range of 2-30 minutes to determine the optimal crosslinking time. Under crosslinking may result in dissociation of the protein-DNA complexes during the ChIP procedure, while crosslinking for too long can mask epitope sites, inhibit chromatin fragmentation, and making reversal of crosslinking more difficult. At the end, glycine (~150 mM final concentration) can be added to quench the crosslinking.

Cell Lysis

Cell lysis is necessary for the release of chromatin from the cell. While whole cell lysis can be performed in one step, isolating nuclei by first removing the cytoplasm tends to result in lower background. As with IP, lysis in ChIP is achieved using buffers containing a mixture of detergents and salts, with the primary aims being to stabilize proteins, inhibit enzyme activity, and rupture membranes to release cellular components.

The cell membrane can be more readily disrupted than the nuclear membrane, meaning weaker, non-ionic detergents such as NP-40 or Triton X-100 can be used to isolate nuclei. This can then be followed by a nuclear lysis step using both mechanical force (e.g. dounce homogenizer, vortexer, or passing the solution through a needle) and a buffer that contains stronger detergents such as sodium dodecyl sulfate (SDS). The inclusion of the crosslinking step in ChIP makes protein-DNA interactions less susceptible to being disrupted by detergents than protein-protein interactions are in co-IP.

In addition to detergents, ChIP lysis buffers tend to contain NaCl and/or Tris-HCl, and usually have a slightly basic pH (7.4 to 8), though this can be optimized (between pH 6-9). Other buffer components that can be optimized include: salts (0-1 M) for maintaining ionic strength and correct tonicity for easy cell lysis; divalent cations such as Mg²⁺ (0-10 mM) which can help to prevent DNA causing the solution to become viscous; EDTA (0-5 mM) for chelating ions such as Zn²⁺ that proteases require for proteolytic function; and enzyme inhibitors, discussed further below.

Successful lysis can easily be checked under a microscope by comparing 10 µl of sample before and after lysis on a hemocytometer, which will confirm the release of nuclei and subsequent nuclear lysis. Cell lysis varies depending on the cell type, so should be optimized by gradually increasing the stringency of buffer components, increasing incubation time, or by using mechanical force. Regardless of the composition of the buffer, performing all steps of a ChIP at 4°C or on ice is strongly recommended in order to minimize sample degradation.

Enzyme Inhibitors

The final components of lysis buffers are inhibitors of proteases and enzymes that alter protein PTMs, particularly phosphatases. Protease inhibitors prevent the degradation of target proteins, while PTMs are often necessary for protein-protein interactions and so must be maintained for co-IP. Preserving PTMs is obviously essential if the target of the IP is to understand the PTM state of target proteins.

Inhibitors should be added fresh, immediately before lysis buffer use. Table 2 and Table 3 contain common inhibitors used in lysis buffers.

Table 2: Commonly used protease inhibitors in co-IP. Note that protease inhibitor cocktails are commercially available (often as tablets) and provide a convenient way to ensure protease inhibition.

Table 3: Commonly used phosphatase Inhibitors in co-IP. Phosphatase inhibitor cocktails are commercially available (often as tablets) and provide a convenient way to ensure phosphatase inhibition. Note that while phosphatase inhibitors are typically included in IP buffers, inhibitors of other PTMs, such as ubiquitination and methylation, are included only as needed, determined by the aims of the experiment.

Chromatin Fragmentation

Chromatin in its native form is high molecular weight, and so must be fragmented to render the chromatin soluble and accessible to the ChIP antibody. Chromatin can be fragmented either by sonication or by enzymatic digestion, with sonication being more common. Shearing by sonication should only be used with X-ChIP, because sonication will disrupt protein-DNA interactions without crosslinking in N-ChIP. Table 4 below shows a comparison between the two approaches.

Table 4: Comparison of major features of X-ChIP and N-ChIP.

Regardless of which approach is taken, chromatin fragmentation must be optimized for each cell type to give fragments of the appropriate length. The ideal fragment length is generally thought to be 200-750 bp, though some studies have found that 150-300 bp (1 to 2 nucleosome units) with an average of 200-250 bp was optimal for ChIP-seq.^27,28 Some of the sample can be examined by agarose gel electrophoresis to determine the optimal fragmentation conditions (Figure 3), and ideally would be performed during each experiment to confirm ideal fragment length. Prior to gel electrophoresis, DNA samples should be cleaned up by reversing the crosslinking (e.g. by heating at 65°C) and treating with proteinase K and RNase A to remove protein and RNA, respectively, that might otherwise interfere. This is then followed by DNA purification using a spin column or phenol-chloroform extraction.

Antibody Selection

Antibodies are generated by the immune system of a host organism (e.g. mouse, rabbit) that has been repeatedly immunized with a specific antigen. The antibodies recognize and specifically bind to that antigen with a high affinity, allowing the protein to be precipitated from the lysate. High specificity is essential for a successful ChIP, as non-specific binding can result in high background or false positive results, and ChIP targets often only differ by small chemical groups such as PTMs. For example, mono-, di and tri-methyl groups on histones can indicate different activity states: H3K9me1 is generally an activating mark, but H3K9me2 is generally a repressive mark.

To confirm the specificity of the antibody-antigen interaction, an isotype control should be included. This control is often called a “mock” IP, and consists of a non-immune antibody of the same isotype as the experimental antibody. This should not have any affinity for the target antigen, but it may non-specifically bind to other factors. Therefore, any DNA sequences that emerge from both the ChIP and the isotype control conditions are unlikely to be specifically bound by the protein of interest.

The following factors should be taken into account when selecting an antibody.

Bead support

The type of beads chosen when performing ChIP is an important consideration and will dictate certain aspects of the overall protocol. Beads are usually either agarose (or Sepharose, which is a tradename for a crosslinked, beaded-form of agarose) or magnetic beads. Table 5 summarizes the key differences between agarose and magnetic beads, which are described further below.

Table 5: Key features of agarose and magnetic beads in ChIP.

Immobilizing Antibodies to Beads

Immobilizing antibodies to the solid phase (beads) is essential for ChIP, enabling the precipitation of the target protein-DNA complexes. While the most common method is to use Protein A or G to capture the ChIP antibody, various approaches can be used, each of which has its own benefits and applications. These approaches are illustrated in Figure 4 and explained in more detail below.

Figure 4: Approaches to IP antibody immobilization on beads.A, Protein A, G or A/G bind to the Fc region of antibodies. B, Antibodies can be crosslinked to Protein A, G or A/G to prevent eluting the antibodies during antigen elution. C, Protein L binds to the light chain of antibodies. D, Direct immobilization of antibodies to beads obviates the need for Protein A, G or L, and prevents antibody elution. E, Streptavidin-conjugated beads capture biotinylated IP antibodies with very high affinity. F, Secondary antibodies directly bound to beads can be used to capture IP antibodies from a specific host or of a specific isotype, but offer less flexibility than Protein A or G.

Pre-clearing and Pre-blocking

Pre-clearing is an optional but often worthwhile step to reduce non-specific binding in ChIP. Pre-clearing refers to incubating samples with the beads (including Protein A/G or other attachment substrates) in order to remove lysate components that bind to bead non-specifically. This prevents these components from being carried through and ultimately eluted, therefore reducing background signal.

Pre-clearing is not necessary if the target is particularly abundant, and is less important if using magnetic beads over agarose beads, because magnetic beads are less prone to non-specific binding.

Prior to pre-clearing, 1-10% of the chromatin should be conserved before the addition of any antibody or beads. This represents the starting material and will be run alongside samples during qPCR or sequencing analysis to normalize ChIP-enriched sequences.

Reducing non-specific binding can also be achieved by pre-blocking the bead. This works in a similar way to blocking in immunostaining, western blotting and ELISA. The bead is incubated with a mild blocking buffer containing 0.1-1% BSA, non-fat milk or 0.1-1% Tween-20 to block sites of non-specific binding on the bead, preventing lysate components from binding. Blocking non-specific DNA binding to beads can be achieved by blocking with salmon sperm DNA, though this is not recommended for ChIP-seq applications because of contaminating reads.

Immunoprecipitation

As in IP and co-IP, there are two approaches for ChIP: the pre-immobilized antibody method in which the antibody is incubated with the bead first, and the free-antibody method in which the antibody is added to the chromatin first before the beads are added. Using free antibodies can be beneficial in instances where the target protein is low abundance as it can give the highest yield, though it can also give higher background than the pre-immobilized approach. The pre-immobilized method must be used when using direct immobilization approaches. Once the approach has been decided upon, the antibodies are added to the sample and incubated overnight at 4°C.

Washing

After the antibodies and beads have been added, the bead-antibody-antigen complexes are pelleted, allowing the supernatant to be removed. The beads then need to be washed to remove non-specific binding of other lysate components to the bead, immobilization substrate or antibody. Washing is usually done using multiple dedicated washing buffers of increasing stringencies, including low salt (NaCl), high salt, and lithium chloride (LiCl) buffers, before a final Tris-EDTA (TE) buffer wash to prepare the DNA for elution.

Other components of the wash buffers include Tris-HCl as a base, containing detergents such as 0.1% SDS and 0.5-1% NP-40 or Triton X-100 to reduce non-specific binding. Salt (NaCl) can be increased up to 1 M to increase the stringency of the wash by reducing ionic and electrostatic interactions, while LiCl provides even higher stringency. These buffers will also contain EDTA to inhibit DNases, as well as protease and phosphatase inhibitors.

Elution and Crosslink Reversal

Elution refers to dissociating the target protein-DNA complex from the beads to obtain DNA for analysis. Elution buffers usually contain sodium carbonate and SDS.

To obtain a pure DNA sample, however, the formaldehyde crosslinks must be reversed and contaminants removed. Heating at 95°C for 15 minutes may be sufficient to reverse crosslinks, but sometimes long heat treatment at 65°C alongside proteinase K treatment is recommended. RNase can also be added to remove contaminating RNA. Finally, the DNA sample should be cleaned up using a phenol-chloroform extraction or with spin columns.

Analysis

The typical aim of ChIP experiments is to identify specific genomic regions that are occupied by a given DNA-binding protein or modified histone protein. qPCR and NGS are the two most common ways of assessing this.

References

Diagrams created with BioRender.com.

1. Barrett, B., Wood, P. A. & Volwiler, W. Quantitation of gamma globulins in human serum by immunoprecipitation. J. Lab. Clin. Med. 55, 605–615 (1960).
2. DeCaprio, J. & Kohl, T. O. Immunoprecipitation. Cold Spring Harb. Protoc. 2020, pdb.top098509 (2020).
3. Gilmour, D. S. & Lis, J. T. Detecting protein-DNA interactions in vivo: distribution of RNA polymerase on specific bacterial genes. Proc. Natl. Acad. Sci. U. S. A. 81, 4275–4279 (1984).
4. Gilmour, D. S. & Lis, J. T. In vivo interactions of RNA polymerase II with genes of Drosophila melanogaster. Mol. Cell. Biol. 5, 2009 (1985).
5. Gilmour, D. S. & Lis, J. T. RNA polymerase II interacts with the promoter region of the noninduced hsp70 gene in Drosophila melanogaster cells. Mol. Cell. Biol. 6, 3984–3989 (1986).
6. Gilmour, D. S., Pflugfelder, G., Wang, J. C. & Lis, J. T. Topoisomerase I interacts with transcribed regions in Drosophila cells. Cell 44, 401–407 (1986).
7. Solomon, M. J., Larsen, P. L. & Varshavsky, A. Mapping protein-DNA interactions in vivo with formaldehyde: Evidence that histone H4 is retained on a highly transcribed gene. Cell 53, 937–947 (1988).
8. Hebbes, T. R., Thorne, A. W. & Crane‐Robinson, C. A direct link between core histone acetylation and transcriptionally active chromatin. EMBO J. 7, 1395–1402 (1988).
9. Mundade, R., Ozer, H. G., Wei, H., Prabhu, L. & Lu, T. Role of ChIP-seq in the discovery of transcription factor binding sites, differential gene regulation mechanism, epigenetic marks and beyond. Cell Cycle 13, 2847–2852 (2014).
10. Blat, Y. & Kleckner, N. Cohesins bind to preferential sites along yeast chromosome III, with differential regulation along arms versus the centric region. Cell 98, 249–259 (1999).
11. Weinmann, A. S., Bartley, S. M., Zhang, T., Zhang, M. Q. & Farnham, P. J. Use of Chromatin Immunoprecipitation To Clone Novel E2F Target Promoters. Mol. Cell. Biol. 21, 6820–6832 (2001).
12. Robertson, G. et al. Genome-wide profiles of STAT1 DNA association using chromatin immunoprecipitation and massively parallel sequencing. Nat. Methods 4, 651–657 (2007).
13. Gibcus, J. H. & Dekker, J. The Hierarchy of the 3D Genome. Mol. Cell 49, 773–782 (2013).
14. Cutter, A. & Hayes, J. J. A Brief Review of Nucleosome Structure. FEBS Lett. 589, 2914–2922 (2015).
15. Luger, K. Structure and dynamic behavior of nucleosomes. Curr. Opin. Genet. Dev. 13, 127–135 (2003).
16. Singh, D., Nishi, K., Khambata, K. & Balasinor, N. H. Introduction to epigenetics: basic concepts and advancements in the field. in Epigenetics and Reproductive Health vol. 21 xxv–xliv (2020).
17. Wang, H. et al. Role of histone H2A ubiquitination in Polycomb silencing. Nature 431, 873–878 (2004).
18. Allshire, R. C. & Madhani, H. D. Ten principles of heterochromatin formation and function. Nat. Rev. Mol. Cell Biol. 19, 229–244 (2018).
19. Morrison, O. & Thakur, J. Molecular Complexes at Euchromatin, Heterochromatin and Centromeric Chromatin. Int. J. Mol. Sci. 22, 6922 (2021).
20. Biswas, S. & Rao, C. M. Epigenetic tools (The Writers, The Readers and The Erasers) and their implications in cancer therapy. Eur. J. Pharmacol. 837, 8–24 (2018).
21. Bannister, A. J. & Kouzarides, T. Regulation of chromatin by histone modifications. Cell Res. 21, 381–395 (2011).
22. Wu, Y.-L. et al. Epigenetic regulation in metabolic diseases: mechanisms and advances in clinical study. Signal Transduct. Target. Ther. 8, 1–27 (2023).
23. Skene, P. J. & Henikoff, S. An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites. eLife 6, e21856 (2017).
24. Kaya-Okur, H. S. et al. CUT&Tag for efficient epigenomic profiling of small samples and single cells. Nat. Commun. 10, 1930 (2019).
25. Gade, P. & Kalvakolanu, D. V. Chromatin Immunoprecipitation Assay as a Tool for Analyzing Transcription Factor Activity. in Transcriptional Regulation: Methods and Protocols, 85–104 (2012).
26. Cotney, J. L. & Noonan, J. P. Chromatin Immunoprecipitation with Fixed Animal Tissues and Preparation for High-Throughput Sequencing. Cold Spring Harb. Protoc. 2015, pdb.prot084848 (2015).
27. Kidder, B. L., Hu, G. & Zhao, K. ChIP-Seq: technical considerations for obtaining high-quality data. Nat. Immunol. 12, 918–922 (2011).
28. Keller, C. A. et al. Effects of sheared chromatin length on ChIP-seq quality and sensitivity. G3 Genes|Genomes|Genetics 11, jkab101 (2021).
29. Hjelm, H., Hjelm, K. & Sjöquist, J. Protein A from Staphylococcus aureus. Its isolation by affinity chromatography and its use as an immunosorbent for isolation of immunoglobulins. FEBS Lett. 28, 73–76 (1972).
30. Björck, L. & Kronvall, G. Purification and some properties of streptococcal protein G, a novel IgG-binding reagent. J. Immunol. Baltim. Md 1950 133, 969–974 (1984).
31. Nilson, B. H., Solomon, A., Björck, L. & Akerström, B. Protein L from Peptostreptococcus magnus binds to the kappa light chain variable domain. J. Biol. Chem. 267, 2234–2239 (1992).