Precise determination of protein-binding locations across the genome is important for understanding gene regulation. Here we describe a genomic mapping method that treats chromatin-immunoprecipitated DNA with exonuclease digestion (ChIP-exo) followed by high-throughput sequencing. This method detects protein-DNA interactions with near base-pair mapping resolution and high signal-to-noise ratio in mammalian neurons.
Identification of specific protein-DNA interactions on the genome is important for understanding gene regulation. Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) is widely used to identify genome-wide binding locations of DNA-binding proteins. However, the ChIP-seq method is limited by its heterogeneity in length of sonicated DNA fragments and non-specific background DNA, resulting in low mapping resolution and uncertainty in DNA-binding sites. To overcome these limitations, the combination of ChIP with exonuclease digestion (ChIP-exo) utilizes 5’ to 3’ exonuclease digestion to trim the heterogeneously sized immunoprecipitated DNA to the protein-DNA crosslinking site. Exonuclease treatment also eliminates non-specific background DNA. The library-prepared and exonuclease-digested DNA can be sent for high-throughput sequencing. The ChIP-exo method allows for near base-pair mapping resolution with greater detection sensitivity and reduced background signal. An optimized ChIP-exo protocol for mammalian cells and next-generation sequencing is described below.
The locations of protein-DNA interactions provide insight into the mechanisms of gene regulation. Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) has been used for a decade to examine genome-wide protein-DNA interactions in living cells1,2. However, the ChIP-seq method is limited by heterogeneity in DNA fragmentation and unbound DNA contamination that lead to low mapping resolution, false positives, missed calls, and non-specific background signal. The combination of ChIP with exonuclease digestion (ChIP-exo) improves upon the ChIP-seq method by trimming ChIP DNA to the protein-DNA crosslinking points, providing near base-pair resolution and a low background signal3,4,5. The greatly improved mapping resolution and low background provided by ChIP-exo allow accurate and comprehensive protein-DNA binding locations to be determined across the genome. ChIP-exo is able to reveal functionally distinct DNA-binding motifs, cooperative interactions between transcription factors (TF), and multiple protein-DNA crosslinking sites in a certain genomic binding location, not detectable by other genomic mapping methods3,4,6,7.
ChIP-exo was initially used in budding yeast to examine the sequence-specific DNA binding of TFs, to study the precise organization of the transcription pre-initiation complex, and sub-nucleosomal structure of individual histones across the genome4,8,9. Since its introduction in 20114, ChIP-exo has been successfully utilized in many other organisms including bacteria, mice, and human cells7,10,11,12,13,14,15,16,17. In 2016, Rhee et al.14 used ChIP-exo in mammalian neurons for the first time to understand how neuronal gene expression was maintained after the downregulation of programming TF Lhx3, which forms a heterodimer complex with another programming TF Isl1. This study showed that in the absence of Lhx3, Isl1 is recruited to new neuronal enhancers bound by Onecut1 TF to maintain gene expression of neuronal effector genes. In this study, ChIP-exo revealed how multiple TFs dynamically recognize cell type and cell stage-specific DNA regulatory elements in a combinatorial manner at near-nucleotide mapping resolution. Other studies also used the ChIP-exo method to understand the interplay between proteins and DNA in other mammalian cell lines. Han et al.7 used ChIP-exo to examine genome-wide organization of GATA1 and TAL1 TFs in mouse erythroid cells using ChIP-exo. This study found that TAL1 is directly recruited to DNA rather than indirectly through protein-protein interactions with GATA1 throughout erythroid differentiation. Recent studies also used ChIP-exo to profile the genome-wide binding locations of CTCF, RNA Polymerase II, and histone marks to study epigenomic and transcriptional mechanisms in human cell lines18,19.
There are several versions of the ChIP-exo protocol available3,5,20. However, these ChIP-exo protocols are difficult to follow for researches who are not familiar with next-generation sequencing library preparation. An excellent version of the ChIP-exo protocol was published with easy-to-follow instructions and a video21, but contained many enzymatic steps that require a significant amount of time to complete. Here we report a new version of the ChIP-exo protocol containing reduced enzymatic steps and incubation times, and explanations for each enzymatic step21. End repair and dA-tailing reactions are combined in a single step using end prep enzyme. The incubation times for index and universal adapter ligation steps are reduced from 2 h to 15 min using a ligation enhancer. The kinase reaction after the index adapter ligation step described in the previous ChIP-exo protocol is removed. Instead, a phosphate group is added during oligo DNA synthesis to one of the 5’ ends of the index adapter (Table 1), which will be used for the lambda exonuclease digestion step. While the previous ChIP-exo protocol used RecJf exonuclease digestion to eliminate single-stranded DNA contaminants, this digestion step is removed here because it is not critical for the quality of the ChIP-exo library. In addition, to purify reverse-crosslinked DNA after ChIP elution, a magnetic beads purification method is used instead of the phenol:chloroform:isoamyl alcohol (PCIA) extraction method. This reduces the incubation time of DNA extraction. Importantly, it removes the majority of adapter dimers formed during index adapter ligation, which may impact the efficiency of ligation-mediated PCR.
The ChIP-exo protocol presented here is optimized for the detection of precise protein-DNA interactions in mammalian neurons differentiated from mouse embryonic stem (ES) cells. Briefly, harvested and crosslinked neuronal cells are lysed, to allow chromatin to be exposed to sonication, then sonicated so that appropriately sized DNA fragments are obtained (Figure 1). Antibody-coated beads are then used to selectively immunoprecipitate fragmented, soluble chromatin to the protein of interest. While the immunoprecipitated DNA is still on the beads, end-repair, ligation of sequencing adapters, fill-in reaction and 5’ to 3’ lambda exonuclease digestion steps are performed. The exonuclease digestion step is what gives ChIP-exo its ultra-high resolution and high signal-to-noise ratio. Lambda exonuclease trims the immunoprecipitated DNA a few base-pairs (bp) from the crosslinking site, thus causing contaminating DNA to be degraded. The exonuclease-treated ChIP DNA is eluted from the antibody-coated beads, protein-DNA crosslinks are reversed, and proteins are degraded. DNA is extracted and denatured to single-stranded ChIP DNA, followed by primer annealing and extension to make double-stranded DNA (dsDNA). Next, ligation of a universal adapter to the exonuclease-treated ends is performed. The resulting DNA is purified, then PCR amplified, gel purified, and subjected to next-generation sequencing.
The ChIP-exo protocol is longer than the ChIP-seq protocol, but is not very technically challenging. Any successfully immunoprecipitated ChIP DNA can be subjected to ChIP-exo, with several additional enzymatic steps. The notable advantages of ChIP-exo, such as ultra-high mapping resolution, a reduced background signal, and decreased false positive and negatives, regarding genomic binding sites, outweigh the time cost.
NOTE: Autoclaved distilled and deionized water (ddH2O) is recommended for making buffers and reaction master mixes. Sections 1−4 describe cell lysis and sonication, sections 5−7 describe chromatin immunoprecipitation (ChIP), sections 8−11 describe enzymatic reactions on beads, sections 12 and 13 describe ChIP elution and DNA purification, and sections 14−19 describe library preparation.
1. Harvesting and crosslinking cells
2. Cell lysis
NOTE: The following steps in this protocol are for approximately 2 x 107 neuronal cells differentiated from mouse ES cells. To break open cells, lysis buffers containing various detergents will be used. Add 50 µL of 1000x complete protease inhibitor (CPI) stock to 50 mL of buffer just prior to use.
3. Sonicating chromatin
NOTE: Keep samples on ice or at 4 °C during this sonication procedure to reduce crosslink reversal.
4. Checking sonication
5. Antibody incubation with beads
NOTE: The following steps in this protocol are for approximately 2 x 107 neuronal cells differentiated from mouse ES cells. Do not freeze and thaw magnetic beads at any point during the ChIP-exo protocol as the beads may crack causing contamination of the sample or the antibody’s performance may be compromised.
6. Chromatin immunoprecipitation (ChIP)
7. ChIP washes
NOTE: Keep samples on ice or at 4 °C to maintain protein-DNA crosslinking during ChIP washes.
8. End repair and dA-tailing reaction on beads
NOTE: Sonication often generates non-blunt ended dsDNA. An end-repair reaction is required to make blunt-ended DNA prior to the dA-tailing reaction followed by the index adapter ligation step. For sticky end DNA ligation with index adapter DNA, dATP is added to the 3’ end of a blunt, dsDNA fragment by the dA-tailing reaction. End prep reaction mix contains dATP.
9. Index adapter ligation on beads
NOTE: The index adapter has 6−10 bases of barcoded index sequences, which are specific to a given sample used for multiplexing multiple samples in high-throughput sequencing. Index adapter DNA sequences are described in Table 1.
10. Fill-in reaction on beads
NOTE: After adapter ligation, there is no phosphodiester bond between the 5’ end of the adapter and the 3’ end of the ChIP DNA. The nick can be repaired by a fill-in reaction.
11. Lambda exonuclease digestion on beads
12. Elution and reverse crosslinking
13. DNA extraction
14. Denaturing, primer annealing and primer extension
15. dA-tailing reaction
NOTE: For sticky end DNA ligation with universal adapter DNA, dATP is added to the 3’ end of blunt, dsDNA by the dA-tailing reaction.
16. Universal adapter ligation
NOTE: The universal adapter includes high-throughput sequencing-specific sequences for DNA sample recognition for sequencing chemistry. The universal adapter DNA sequences are described in Table 1.
17. DNA cleanup
18. Ligation-mediated PCR
NOTE: LM-PCR primer sequences are described in Table 1.
19. DNA purification of LM-PCR amplified DNA
Figure 2A illustrates sonication results after cell lysis and sonication, with various cycles, of motor neuron cells differentiated from mouse ES cells. The optimal number of sonication cycles (for example, 12 cycles in Figure 2A) generated strong DNA intensity in 100−400 bp DNA fragments. High-quality ChIP-exo libraries are based on the size and quantity of fragmented chromatin DNA. Thus, optimization of sonication is recommended for each cell type and batch of cells before starting ChIP-exo.
Figure 2B shows ligation-mediated PCR (LM-PCR) products of the ChIP-exo DNA samples amplified by 18−21 PCR cycles. LM-PCR amplifies ChIP-exo DNA fragments that are ligated with DNA adapters for next-generation sequencing. PCR primer is a part of the DNA adapter sequences. The size of sonicated DNA fragments is around 100−400 bp (Figure 2A). After lambda exonuclease digestion, the size of DNA fragments would become the half size of the starting material (50−200 bp). Approximately 125 bp of DNA adapters were ligated to the ChIP-exo DNA fragments thus, the expected size of LM-PCR products is 175−325 bp. The minimal number of PCR cyclesc while still being sufficient to amplify the ChIP-exo library, are performed to avoid over-amplification of ChIP-exo DNA.
Here, ChIP-exo for Isl1 was performed in mouse ES cell-derived motor neurons. Isl1 is a motor neuron programming transcription factor, which is specifically expressed in postmitotic motor neurons. The results for Isl1 ChIP-exo show significant amounts of 200−400 bp LM-PCR amplified ChIP-exo DNA (Figure 2B). The band around 100 bp indicates PCR artifacts from adapters and PCR primers. Running a no antibody control alongside an experimental sample is also important to ensure non-specific, background DNA was digested by the lambda exonuclease treatment. As an example, we identified Isl1 bound locations in mouse ES cell-derived motor neurons using ChIP-exo and ChIP-seq (Figure 3). The ChIP-exo signal was highly focused at Isl1-binding sites, detecting multiple clustered Isl1 transcription factor binding patterns. The ChIP-seq signal displayed broader signals, indicating that ChIP-exo of Isl1 has higher mapping resolution than ChIP-seq of Isl1.
Figure 1: Schematic of the ChIP-exo protocol. After ChIP (steps 1−7), the end-repair and dA-tailing reactions make ChIP DNA blunt-ended by adding a phosphate group to the 5’ end of the DNA and adding dATP to the 3’ end of the DNA (step 8). Sonicated ends of ChIP DNA, on the beads are ligated with the index adapter (step 9). After the fill-in reaction, the adapter DNA with the 5’ overhang becomes blunt-ended (step 10). Lambda exonuclease digests the sonicated DNA 5’ to 3’ up to the protein (TF)-DNA crosslinking points (step 11). After elution, reverse-crosslinking and DNA extraction (steps 12 and 13), denatured single-stranded DNA is made double-stranded by index PCR primer extension (step 14), followed by dA-tailing (step 15). The universal adapter is then ligated to the exonuclease-treated end (step 16). The resulting library is cleaned up, PCR-amplified, and subjected to next-generation sequencing (steps 17−19). Mapping the 5’ ends of the resulting sequencing tags to the reference genome demarcates the exonuclease barrier and thus the precise site of protein-DNA crosslinking.
Figure 2: Sonication and LM-PCR amplification of a ChIP-exo library. (A) 1.5% agarose gel of the electrophoresed sonicated DNA. 12, 18, and 24 cycles of sonication were conducted to find optimal sonication cycles for 2 x 107 cells of mouse ES cell-derived motor neurons. Following sonication, the DNA sample was reverse crosslinked, and extracted using PCIA and ethanol precipitation. Each sample contained 4 x 105 cell equivalents, which is 2% of sonicated cells. 12 cycles of sonication produced a greater yield of sonicated DNA with the desired size (100−500 bp). (B) 1.5% agarose gel of electrophoresed ChIP-exo libraries following 18−21 cycles of LM-PCR for no antibody control (No Ab) and Isl1 in mouse ES cell-derived motor neurons. Each sample contained 10 x 106 cell equivalent of mouse motor neurons. The no antibody ChIP-exo result (lane 2) demonstrates that non-specific, contaminating DNA is eliminated by lambda exonuclease. The ChIP-exo libraries for Isl1 (lane 3) show amplified DNA libraries around 200−400 bp, indicating the ChIP-exo libraries were successfully amplified by adapter ligation-mediated PCR. Please click here to view a larger version of this figure.
Figure 3: Comparison of ChIP-exo to ChIP-seq for Isl1 at specific loci. The blue and red filled plots show the distribution of sequencing tags for ChIP-seq and ChIP-exo Isl1-bound locations, respectively, proximal to Slit3 and Fgfr1 gene in nascent spinal motor neurons differentiated from mouse ES cells.
Table 1: Oligonucleotides used in this protocol.
Oligo name | Length (nt) | Sequence | Note | |||||
Index adapter-forward* | 66 | 5'/Phos/CAAGCAGAAGACGGCATACGAGATXXXXXXXXGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT 3' | 5' phosphate, index (XXXXXXXX), 3' T-overhang | |||||
Index adapter-reverse* | 33 | 5'GATCGGAAGAGCACACGTCTGAACTCCAGTCAC 3' | ||||||
Ligation adapter-forward* | 58 | 5'AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT 3' | ||||||
Ligation adapter-reverse* | 34 | 5'GATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAG 3' | ||||||
Index PCR primer* | 22 | 5'CAAGCAGAAGACGGCATACGAG 3' | ||||||
Universal PCR primer* | 19 | 5'AATGATACGGCGACCACCG 3' | ||||||
*The above sequencing adapters and PCR primers are designed for Illumina HiSeq and NextSeq Sequencing Platforms. |
Table 2: Recipes for lysis buffers 1−3 and blocking buffer. Store in 50 mL tubes at 4 °C. Add 50 µL of 1000x CPI (complete protease inhibitor) stock to all buffers just prior to use.
Lysis buffer 1 | ||
Reagent | Volume (mL) | [Final] |
1 M HEPES (pH 7.3) | 2.5 | 50 mM |
5 M NaCl | 1.4 | 140 mM |
0.5 M EDTA (pH 8.0) | 0.1 | 1 mM |
50% Glycerol | 10 | 10% |
10% Octylphenol ethoxylate | 2.5 | 0.50% |
10% 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol | 1.25 | 0.25% |
Autoclaved ddH2O | Fill to 50 | |
Lysis buffer 2 | ||
Reagent | Volume (mL) | [Final] |
0.5 M Tris-HCl (pH 8.0) | 1 | 10 mM |
5 M NaCl | 2 | 200 mM |
0.5 M EDTA (pH 8.0) | 0.1 | 1 mM |
Autoclaved ddH2O | Fill to 50 | |
Lysis buffer 3 | ||
Reagent | Volume (mL) | [Final] |
1 M Tris-HCl (pH 8.0) | 0.5 | 10 mM |
5 M NaCl | 1 | 100 mM |
0.5 M EDTA (pH 8.0) | 0.1 | 1 mM |
10% Deoxycholic Acid | 0.5 | 0.10% |
30% N-Lauroylsarcosine sodium salt solution | 0.83 | 0.50% |
Autoclaved ddH2O | Fill to 50 | |
Blocking solution | ||
Reagent | Amount | [Final] |
Bovine serum albumin (BSA) | 250 mg | 0.50% |
Complete Protease Inhibitor (CPI, 1000x) | 50 µL | 1x |
Phosphate buffered saline (PBS) | Fill to 50 mL |
Table 3: Recipes for ChIP washes: High Salt Wash buffer, LiCl Wash buffer and 10 mM Tris-HCl buffer (pH 7.4). Store in 50 mL tubes at 4 °C. Add 50 µL of 1000x CPI stock to all buffers just prior to use.
High salt wash buffer | ||
Reagent | Volume (mL) | [Final] |
1 M HEPES (pH 7.3) | 2.5 | 50 mM |
5 M NaCl | 5 | 500 mM |
0.5 M EDTA (pH 8.0) | 0.1 | 1 mM |
10% 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol | 5 | 1% |
5% Deoxycholic acid | 1 | 0.10% |
5% SDS | 1 | 0.10% |
Autoclaved ddH2O | Fill to 50 | |
LiCl wash buffer | ||
Reagent | Volume (mL) | [Final] |
0.5 M Tris-HCl (pH 8.0) | 2 | 20 mM |
0.5 M EDTA (pH 8.0) | 0.1 | 1 mM |
1 M LiCl | 12.5 | 250 mM |
10% Octylphenol ethoxylate | 2.5 | 0.50% |
5% Deoxycholic acid | 5 | 0.50% |
Autoclaved ddH2O | Fill to 50 | |
10 mM Tris-HCl buffer | ||
Reagent | Volume (mL) | [Final] |
1 M Tris-HCl (pH 7.4) | 0.5 | 10 mM |
Autoclaved ddH2O | Fill to 50 |
Table 4: Fill-in reaction master mix.
Reagent | 1x (µL) | [Final] |
20 mg/mL BSA | 0.6 | 0.2 mg/mL |
10x phi29 DNA polymerase buffer | 6 | 1x |
3 mM dNTPs | 2.5 | 150 µM |
10 U/µL phi29 DNA polymerase | 2 | 10 U |
Reaction mix volume | 11.1 |
Table 5: Recipe for ChIP Elution buffer. Store in 50 mL tubes at RT.
Reagent | Volume (mL) | [Final] |
0.5 M Tris-HCl (pH 8.0) | 5 | 50 mM |
0.5 M EDTA (pH 8.0) | 1 | 10 mM |
10% SDS | 5 | 1% |
Autoclaved ddH2O | up to 50 mL |
Table 6: Denaturing and Primer Annealing reaction master mix and program.
Denaturing and primer annealing mix | ||
Reagent | 1x (µL) | [Final] |
20 mg/mL BSA | 0.2 | 0.2 mg/mL |
3 mM dNTPs | 0.5 | 75 µM |
10 µM Index PCR primer | 0.5 | 0.25 µM |
Reaction mix volume | 1.2 | |
Denaturing and primer annealing program | ||
Temperature (°C) | Time | |
95 | 5 min | |
65 | 3 min | |
60 | 3 min | |
57 | 3 min | |
54 | 3 min | |
51 | 3 min | |
30 | Forever |
Table 7: Primer Extension reaction master mix and program.
Primer extension mix | ||
Reagent | 1x (µL) | [Final] |
10x phi29 DNA polymerase buffer | 2 | 1x |
10 U/µL phi29 DNA polymerase | 1 | 75 µM |
Reaction mix volume | 3 | |
Primer extension program | ||
Temperature (°C) | Time | |
30 | 20 min | |
65 | 10 min | |
4 | Forever |
Table 8: dA-Tailing reaction master mix and program.
dA-Tailing mix | ||
Reagent | 1x (µL) | [Final] |
3 mM dATP | 0.7 | 120 µM |
Klenow fragment buffer | 2.4 | 1x |
5 U/µL Klenow fragment | 1 | 5 U |
Reaction mix volume | 4.1 | |
dA-Tailing program | ||
Temperature (°C) | Time | |
37 | 30 min | |
75 | 20 min | |
4 | Forever |
Table 9: Universal Adapter Ligation reaction master mix.
Reagent | 1x (µL) | [Final] |
Autoclaved water | 5 | |
15 µM Universal adapter* | 1 | 320 nM |
Ligation enhancer | 0.5 | 1x |
Ligase master mix | 15 | 1x |
Reaction mix volume | 21.5 | |
*Universal adapter DNA sequence is described in Table 1. |
Table 10: Ligation-Mediated PCR master mix and program.
LM-PCR mix | ||
Reagent | 1x (µL) | [Final] |
10 µM Index PCR primer* | 2 | 0.2 µM |
10 µM Universal PCR primer* | 2 | 0.2 µM |
2x Taq DNA polymerase PCR master mix | 25 | 1x |
Reaction mix volume | 29 | |
*PCR primer sequences are described in Table 1. | ||
LM-PCR program | ||
Temperature (°C) | Time | Cycles |
98 | 30 s | 1 |
98 | 10 s | 15−25 |
65 | 75 s | |
65 | 5 min | 1 |
4 | Hold |
In this protocol, ChIP followed by exonuclease digestion is used to obtain DNA libraries for the identification of protein-DNA interactions in mammalian cells at ultra-high mapping resolution. Many variables contribute to the quality of the ChIP-exo experiment. Critical experimental parameters include the quality of antibodies, optimization of sonication, and the number of LM-PCR cycles. These critical experimental parameters are also what can limit ChIP-exo experiments and will be discussed below.
In any ChIP protocol, antibody quality is one of the most important considerations. The use of ChIP-grade antibodies is recommended for ChIP-exo. The antibody quality can be checked prior to conducting the ChIP-exo protocol. ChIP-seq or ChIP-qPCR can be used to validate ChIP-grade antibodies by confirming specific DNA binding locations of the protein of interest. Subsequently, optimizing the concentration of antibodies added to the beads is ideal to ensure that protein-DNA complexes are immunoprecipitated22,23.
The sonication of chromatin is another critical step in the ChIP-exo protocol. Sonication is critical for the non-specific shearing of chromatin, necessary for optimal immunoprecipitation to the protein of interest24. The size and amount of sonicated DNA are dependent on the number of sonication cycles. A high concentration of fragmented DNA is important to ensure that enough DNA is immunoprecipitated to the protein of interest for a ChIP-exo DNA library to be created. Obtaining 100−500 bp fragmented DNA is ideal because the resolution of ChIP-exo is better if the sonicated chromatin fragments have smaller starting sizes. Therefore, the optimal chromatin sonication should be determined for each type and batch of cells by varying the number of sonication cycles and sonication buffers.
The final critical parameter is obtaining the amplification of ChIP-exo library DNA with the minimal number of LM-PCR cycles to avoid over-amplification of PCR artifacts. To determine the minimal number of PCR cycles to amplify ChIP-exo DNA, a small portion of ChIP-exo DNA can be used to run multiple PCR cycles (for example, 10, 15, and 20 cycles) and compare the PCR products.
ChIP-exo has many more steps than a traditional ChIP-seq and, as a result, each step should be performed carefully and precisely for the ChIP-exo protocol to work. Importantly, the correct volume of every reagent in enzymatic reactions must be added to each reaction master mix21. Thus, creating a spreadsheet to calculate the volume of each reagent required for each master mix, then printing the tables and checking each reagent after addition to the master mixes is recommended. Careful excision of the amplified DNA is also important; fragments below 200 bp often contain adapter dimers, which need to be removed before next-generation sequencing.
Notably, most of the critical parameters and limitations of ChIP-exo are identical to those of ChIP-seq. However, unlike ChIP-seq, ChIP-exo delivers high-resolution genome mapping with low background. Thus, ChIP-exo is the ideal method for identifying precise protein-DNA interactions in a variety of systems and organisms, helping to reveal the significant roles of DNA-binding proteins in the cell. The ChIP-exo method described above can be used to examine transcription factors, histone modification marks and chromatin regulatory proteins in living cells at higher mapping resolution than ChIP-seq25,26,27. In addition, ChIP-exo can detect the individual binding locations of DNA-binding proteins within a cluster, while ChIP-seq cannot due to its mapping resolution. Importantly, ChIP-exo displays a higher signal-to-noise ratio compared to ChIP-seq. These advantages of ChIP-exo allow us to identify a comprehensive set of bound locations across the genome. This protocol will provide a foundation for researchers interested in examining DNA-binding locations of various proteins across the genome at near base-pair resolution.
The authors have nothing to disclose.
We thank the member of the Rhee laboratory for sharing unpublished data and valuable discussions. This work was supported by Natural Sciences and Engineering Research Council of Canada (NSERC) grant RGPIN-2018-06404 (H.R.).
Agarose, UltraPure | Invitrogen | 16500 | Checking Sonication (Section 4.3.6) and Gel Purification of LM-PCR Amplified DNA (Section 19.1) |
Albumin, Bovine Serum (BSA), Protease Free, Heat Shock Isolation, Min. 98% | BioShop | ALB003 | Blocking Solution |
Antibody against Isl1 | DSHB | 39.3F7 | Antibody incuation with beads (Section 5.6) |
Bioruptor Pico | Diagenode | B01060010 | Sonicating Chromatin (Section 3.3) |
Bovine serum albumin (BSA), Molecular Biology Grade | New England BioLabs | B9000S | Fill-in Reaction on Beads (Section 10.2) and Denaturing, Primer Annealing and Primer Extension (Section 14.2) |
Centrifuge 5424 R | Eppendorf | 5404000138 | Sonicating Chromatin (Section 3.5), Checking Sonication (Section 4.3.1, 4.3.3 and 4.3.4) |
Centrifuge 5804 R | Eppendorf | 22623508 | Harvest, cross-linking and freezing cells (Section 1.3), Cell lysis (Section 2.2 and 2.3), Sonicating Chromatin (Section 3.1) |
cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail | Roche | 4693159001 | Added to all buffers, except Proteinase K buffer and ChIP Elution buffer |
dATP Solution | New England BioLabs | N0440S | dA-Tailing Reaction (Section 15.1) |
Deoxycholic Acid Sodium Salt | fisher scientific | BP349 | Lysis Buffer 3, High Salt Wash Buffer and LiCl Wash Buffer |
dNTP Mix, Molecular Biology Grade | Thermo Scientific | R0192 | Fill-in Reaction on Beads (Section 10.2) and Denaturing, Primer Annealing and Primer Extension (Section 14.2) |
DreamTaq Green PCR Master Mix, 2x | Thermo Scientific | K1081 | Ligation-Mediated PCR (Section 18.1) |
EDTA, 0.5 M, Sterile Solution, pH 8.0 | BioShop | EDT111 | Lysis Buffer 1-3, Checking Sonication (Section 4.1), High Salt Wash Buffer, LiCl Wash Buffer and ChIP Elution Buffer |
Ethyl Alcohol Anhydrous, 100% | Commercial alcohols | P006EAAN | Checking Sonication (Section 4.3.3) |
Formaldehyde, 36.5-38%, contains 10-15% methanol | Sigma | F8775 | Harvest, cross-linking and freezing cells (Section 1.1) |
Glycerol, Reagent Grade, min 99.5% | BioShop | GLY002 | Lysis Buffer 1 |
Glycine, Biotechnology Grade, min. 99% | BioShop | GLN001 | Harvest, cross-linking and freezing cells (Section 1.2) |
Glycogen, RNA Grade | Thermo Scientific | R0551 | Checking Sonication (Section 4.3.3) |
HEPES, 1 M Sterile-filtered Solution, pH 7.3 | BioShop | HEP003 | Lysis Buffer 1, High Salt Wash Buffer |
Klenow Fragment (3->5 exo-) | New England BioLabs | M0212S | dA-Tailing Reaction (Section 15.1) |
Lambda exonuclease | New England BioLabs | M0262S | Lambda Exonuclease Digestion on Beads (Section 11.2) |
Ligase Enhancer | New England BioLabs | E7645S | NEBNext Ultra II DNA Library Kit. Index Adapter Ligation on Beads (Section 9.1) and Universal Adapter Ligation (Section 16.1) |
Ligase Master Mix | New England BioLabs | E7645S | NEBNext Ultra II DNA Library Kit. Index Adapter Ligation on Beads (Section 9.1) and Universal Adapter Ligation (Section 16.1) |
Lithium Chloride (LiCl), Reagent grade | Bioshop | LIT704 | LiCl Wash Buffer |
Magnetic beads for ChIP (Dynabeads Protein G) | Dynabeads Protein G (magnetic beads for ChIP) | Dynabeads Protein G (magnetic beads for ChIP) | Antibody incubation with beads (Section 5) |
Magnetic beads for DNA purification (AMPure XP Beads) | Beckman Coulter | A63880 | DNA Extraction (Section 13.3) and DNA Clean-up (Section 17.1) |
Magnetic rack (DynaMag-2 Magnet) | Invitrogen | 12321D | Used in many steps in Sections: 5 – 11, 13 |
MinElute Gel Extraction Kit | Qiagen | 28604 | Gel Purification of PCR Amplified DNA (Section 19.2) |
N-Lauroylsarcosine sodium salt solution, 30% aqueous solution, ≥97.0% (HPLC) | Sigma | 61747 | Lysis Buffer 3 |
Octylphenol Ethoxylate (IGEPAL CA630) | BioShop | NON999 | Lysis Buffer 1 and LiCl Wash Buffer |
Phenol:Chloroform:Isoamyl Alcohol, Biotechnology Grade (25:24:1) | BioShop | PHE512 | Checking Sonication (Section 4.3.1) |
phi29 DNA Polymerase | New England BioLabs | M0269L | Fill-in Reaction on Beads (Section 10.2) and Denaturing, Primer Annealing and Primer Extension (Section 14.3 and 14.4) |
Phosphate-Buffered Saline (PBS), 1x | Corning | 21040CV | Harvest, cross-linking and freezing cells (Section 1.3) and Sonicating Chromatin (Section 3.1), Antibody incuation with beads (Section 5.1) |
PowerPac Basic Power Supply | BioRad | 1645050 | Checking Sonication (Section 4.3.6) and Gel Purification of LM-PCR Amplified DNA (Section 19.1) |
ProFlex PCR System | Applied Biosystems | ProFlex PCR System | Used in Sections: 14.2, 14.4, 15.2, 16.2 and 18.2 |
Protein LoBind Tube, 2.0 mL | Eppendorf | 22431102 | Antibody Incubation with Beads (Section 5.2) and Chromatin Immunoprecipitation (Section 6.3) |
Proteinase K Solution, RNA Grade | Invitrogen | 25530049 | Checking Sonication (Section 4.2) and Elution and Reverse Crosslinking (Section 12.3) |
Qubit 4.0 Fluorometer | Invitrogen | Q33226 | Gel Purification of PCR Amplified DNA (Section 19.3) |
Quibit dsDNA BR assay kit | Invitrogen | Q32853 | Gel Purification of PCR Amplified DNA (Section 19.3) |
Rnase A, Dnase and Protease-free | Thermo Scientific | EN0531 | Checking Sonication (Section 4.3.2) and DNA Extraction (Section 13.2) |
Sodium chloride (NaCl), BioReagent | Sigma | S5886 | Lysis Buffer 1-3, High Salt Wash Buffer |
Sodium Dodecyl Sulfate (SDS), Electrophoresis Grade | BioShop | SDS001 | Checking Sonication (Section 4.1), High Salt Wash Buffer and ChIP Elution Buffer |
Sonication beads and 15 mL Bioruptor Tubes | Diagenode | C01020031 | Sonicating Chromatin (Section 3.1 and 3.2) |
ThermoMixer F1.5 | Eppendorf | 5384000020 | Section 4.2, 4.3.2, 4.3.5, 8.2, 9.2, 10.3, 11.2, 12.2, 12.3, 13.2 and 16.2 |
Trizma hydrochloride solution (Tris-HCl), BioPerformance Certified, 1 M, pH 7.4 | Sigma | T2194 | 10 mM Tris-HCl Buffer |
Trizma hydrochloride solution (Tris-HCl), BioPerformance Certified, 1 M, pH 8.0 | Sigma | T2694 | Lysis Buffer 2, Lysis Buffer 3, Checking Sonication (Section 4.1), LiCl Wash Buffer and ChIP Elution Buffer |
Ultra II End Repair/dA-Tailing Module (24 rxn -> 120 rxn) | New England BioLabs | E7546S | End Prep Reaction mix and End Prep Enzyme mix. End-repair and dA-Tailing Reaction on Beads (Section 8.2) |
VWR Mini Tube Rocker, Variable Speed | VWR | 10159-752 | Used in many steps in sections: 1, 2, 5, 6 and 7 |
2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol, Triton X-100, Reagent Grade | BioShop | TRX506 | Lysis Buffer 1, Sonicating Chromatin (Section 3.4) and High Salt Wash Buffer |