Genome-wide Interrogation of Protein-DNA Interactions in Mammalian Cells Using ChIPmentation

Summary Mapping the genomic locations of chromatin-associated proteins, such as transcription factors and histone modifications, is key to understanding the mechanisms of transcriptional regulation. ChIPmentation offers a simple and robust way of investigating the genomic binding sites of a protein using relatively low-input material. Here, we present a detailed protocol for the key steps that lead to a successful ChIPmentation experiment, as well as a quick analysis pipeline to examine the data. For complete details on the use and execution of this protocol, please refer to Schmidl et al. (2015). For example data produced by this protocol, please refer to Henriksson et al. (2019) and Zhang et al. (2019).


SUMMARY
Mapping the genomic locations of chromatin-associated proteins, such as transcription factors and histone modifications, is key to understanding the mechanisms of transcriptional regulation. ChIPmentation offers a simple and robust way of investigating the genomic binding sites of a protein using relatively low-input material. Here, we present a detailed protocol for the key steps that lead to a successful ChIPmentation experiment, as well as a quick analysis pipeline to examine the data. For complete details on the use and execution of this protocol, please refer to Schmidl et al. (2015). For example data produced by this protocol, please refer to Henriksson et al. (2019) and Zhang et al. (2019).

BEFORE YOU BEGIN
There are many existing ChIP-seq protocols with different modifications. Many of them are lengthy and difficult to carry out. We have found ChIPmentation is the simplest and the most robust to implement in a molecular biology lab, especially for people who have already had the experience with the ChIP technique. The ChIPmentation method is modular, where it contains a ChIP module and a library preparation module. Experienced researchers can just stick to their own ChIP protocol and start following the procedures described here after washing the IP (i.e., step 13). People with no previous ChIP experience are recommended to follow the exact procedures described in this protocol. It is also recommended to read the full protocol before starting in order to get a feeling about the timing and work load in each step.
Compared to other ChIP-seq methods, one advantage of ChIPmentation is its sensitivity. We routinely use 5 3 10 5 cells to profile histone modifications and 5 3 10 6 cells to study transcription factors. The minimum cell number required for a successful ChIPmentation experiment in our hands is 10 4 cells for histone modifications and 10 5 cells for transcription factors. However, it is worth noting that the number of cells required for a successful ChIPmentation experiment depends on many factors, such as the abundance of the protein/modification of interest and the efficiency of the antibody. The other major advantage of ChIPmentation is simplicity. Sequencing adapters are added by the transposase Tn5, and library PCR is performed immediately after reverse crosslinking and DNA purification. The third advantage is the cost. Only a small amount of Tn5 transposase is needed per library. Nowadays, we always use ChIPmentation even when the cell number is not a constraint, such as cell lines.

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Note: It is very important to find the right condition for sonication and a good antibody for the immunoprecipitation of the protein of your interest. In general, one can refer to the ENCODE and modENCODE guidelines (Landt et al., 2012). This section describes some extra details on how to check sonication and perform an initial test on antibodies. Sonication serves two purposes: to solubilize chromatin and to fragment DNA to a size range that is suitable for next generation sequencing. However, during the sonication process, we found the epitopes of some proteins can be destroyed as well. The main purpose here is to find a balance between getting the DNA to the right range and maintain the protein integrity at the same time. This is the most variable process in the entire protocol, because sonication is highly dependent on the machine in use. We have successful experience using both probe sonicators and water bath sonicators.
4. After resuspending cell pellet at a concentration of 5 3 10 6 cells per 300 mL Sonication/IP Buffer (i.e., step 6): Note: both the cell concentration and the volume can influence sonication results. We routinely used 300 mL Sonication/IP Buffer to resuspend cells from 10 5 to 5 3 10 6 . When more than 5 3 10 6 cells are used, we scale up the volume to maintain a concentration of 5 3 10 6 per 300 mL volume. If the final volume exceeds the recommendation of the sonicator, make aliquots to perform sonication. a. Take out 25 mL lysate and mix with 75 mL ChIP Elution Buffer and 1 mL Proteinase K (20 mg/mL). Leave the reaction on a thermomixer at 65 C, with shaking at 1,400 rpm for at least 6 h (or overnight, 12-16 h) for the reverse crosslink. This is the no sonication DNA input. b. Take out 32 mL lysate and mix with 8 mL 53 SDS Loading Buffer. Boil at 99 C for 10 min. This is the no sonication protein input.
5. If using a water bath sonicator, aliquot 300 mL lysate into 1.5 mL Eppendorf tubes (or other tubes required by the sonicator manufacturer). Perform a sonication time course with a recommended ON/OFF cycle setting. For example, if using a Bioruptor Pico, a time course of 2, 4, 6, 8 min with 30 s ON/30 s OFF can be used for an initial trial. At the end of each time course: a. Take out 25 mL lysate from an aliquot and mix with 75 mL ChIP Elution Buffer and 1 mL Proteinase K (20 mg/mL). Leave the reaction on a thermomixer at 65 C, with shaking at 1,400 rpm for at least 6 h (or overnight, 12-20 h) for the reverse crosslink. This is for the DNA size check. b. Take out 32 mL lysate and mix with 8 mL 53 SDS Loading Buffer. Boil at 99 C for 10 min. This is for the protein check.
Note: When using the Bioruptor water bath sonicator, the maximum recommended volume in a 1.5 mL Eppendorf tube is 300 mL. Therefore, it is not possible to just use one tube for the entire time course due the volume needed for the DNA and protein analysis. We normally prepare multiple tubes, one for each time point.
6. Analyze all the protein samples using western blot with the antibody of your choice. We include in the Key Resources Table the antibodies used in this particular protocol, but the antibodies will depend on the factors of interest. Four examples are shown in Figure 1.
CRITICAL: This is the initial test of the antibody and protein integrity. There are two things to check here. First, in the no sonication input (0 min), a single (or major) clear band around the predicted size of the protein of interest is present. In Figure 1, all four antibodies satisfy this standard. Second, the protein of interest remains detectable during sonication. In Figure 1 next step to decide which condition to use. For some lowly expressed proteins, more concentrated cell lysate needs to be used to visualize them on the western blot.
7. Purify all the DNA samples after the reverse crosslink using the Qiagen minElute PCR Purification Kit. Determine the DNA concentration using a Nanodrop. Run equal amount of DNA (we routinely use 500-1,000 ng) on a 1.5% Agarose gel. An example is shown in Figure 2.
CRITICAL: Normally one should choose the minimum sonication time that gives rise to the ideal range (100-500 bp). However, the results from the western blot and DNA gel need to be considered together to make a decision. An earliest condition where the DNA is sheared and the protein is still detectable should be chosen. ChIPmentation will use Tn5 to ''cut and paste'' the sequencing adapters to the DNA after the immunoprecipitation, which results in the fragmentation of DNA for a second time. Therefore, it is generally okay to have slightly larger DNA fragments comparing to the traditional ChIP-seq method at this stage. As long as the majority of the DNA is below 1,000 bp and there is no clear band above 1,000 bp, we accept the condition. In this case, Lane 3 is chosen. See Troubleshooting 1 for some tips.

Antibody Test II
Timing: 3 days or more

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In the previous section, the sonication condition is determined and whether the antibody is able to detect the protein of interest is also tested. In this section, procedures are described to test if the antibody can immunoprecipitate the protein of interest after formaldehyde crosslinking. The success of antibody in this test does not necessarily guarantee a successful ChIPmentation experiment, but it provides some useful information about the antibody and the immunoprecipitation condition.
8. Follow steps 1-16 from the Step-by-Step Method Details. a. At the step 6, take 32 mL lysate and mix with 8 mL 53 SDS Loading Buffer. Boil at 99 C for 10 min. This is the input sample and can be stored in À20 C and will be used in the next day. b. At the step 8, before washing, put the immunoprecipitation on the magnetic stand, and take out 32 mL lysate and mix with 8 mL 53 SDS Loading Buffer. Boil at 99 C for 10 min. This is the supernatant. c. After the step 16, instead of adding tagmentation mix, add 40 mL 13 SDS Loading Buffer (diluted in Sonication/IP Buffer from 53 SDS Loading Buffer) to the beads. Boil at 99 C for 10 min. This is the IP sample. 9. Analyze all the samples (input, supernatant and IP) using western blot with the same antibody. An example using an anti-FOXM1 antibody is shown in Figure 3. Figure 3 shown above, 1 mg of anti-FOXM1 antibody was used to immunoprecipitate the chromatin from 5 3 10 6 U2OS cells. When a different antibody is used, different ratios of antibody:chromatin input need to be tested to find a good condition to achieve the result in Figure 3. There are three things to check here. First, a single (or major) clear band around the predicted size of the protein of interest should be present in the input lane. Second, a single (or major) clear band at the same size as the input should be present in the IP lane. One can roughly calculate the IP efficiency based on the intensities of the protein bands, but we generally found the efficiencies of many transcription factor antibodies are low. Nevertheless, they produce successful ChIPmentation results. Third, no or very minimum of the protein of interest or IgG band is visible in the supernatant lane. If the protein of interest is clearly visible in the supernatant, it means the antibody fails to pull down the protein. Whenever this happens, we also observe IgG bands in the supernatant at the same time. This mostly happens with some mouse IgG antibodies that have low affinity to Protein G. We have found the most efficient ways of solving this problem is to either chemically crosslink the antibody to the beads using DSP (or the like) or change to a different type of beads, such as the Pan Mouse IgG Dynabeads. An example using a mouse monoclonal anti-V5 antibody to pull down V5-tagged FOXM1 is shown in Figure 4 to demonstrate this critical point. working with tissues, use an appropriate method to dissociate the tissue into single-cell suspension and start the protocol from step 2b. We normally use 5 3 10 5 cells for profiling histone modifications and 5 3 10 6 cells for transcription factors.

KEY RESOURCES
1. Add 1/10 volume of 11% formaldehyde solution directly to the culture media in plates. Swirl briefly and incubate at room temperature (20 C-25 C) for 10 min. 2. Add 1/10 volume of 1.25 M Glycine, swirl briefly and incubate at room temperature (20 C-25 C) for 5 min to stop formaldehyde crosslinking. a. For adherent cells, remove all liquid in the plate, and rinse with ice-cold 13 PBS (pH 7.4) twice. Collect cells into 1 mL ice-cold 13 PBS (pH 7.4) supplied with 1% Fetal Bovine Serum (FBS) using a cell scraper, and transfer to 1.5 mL Eppendorf tubes. Spin at 4 C for 5 min at 1,000 3 g, and discard supernatant. b. For suspension cells: i. Transfer enough cells into either Eppendorf tubes or conical tubes. If no serum is in the culture media, add 1/100 volume of FBS. ii. Spin at 4 C for 5 min at 1,000 3 g, and discard supernatant. iii. Resuspend cell pellet with the same amount of ice-cold 13 PBS (pH 7.4), spin at 4 C for 5 min at 1,000 3 g, and discard supernatant. Repeat once.
CRITICAL: The addition of FBS during those steps help reduce the loss of cells, especially when the cell number is small.
3. Cell pellets can be snap freezed in liquid nitrogen and store in À80 C for at least 6 months. Or cell pellets can be used immediately. See the next section. 4. Bind antibodies to Dynabeads. We use 10 mL Dynabeads and 1 mg antibody per ChIP. Protein A or G Dynabeads, or Pan Mouse IgG Dynabeads are chosen based on the primary antibody being used and the results from the previous section. Prepare each ChIP individually in different Eppendorf tubes. a. Mix 10 mL Dynabeads with 500 mL Blocking Solution in the tube, and collect beads using DynaMag-2. Allow beads to set at the side of the tube. Invert twice or three times to collect beads at the tube cap. No need to centrifuge. Remove the supernatant with an aspirator. b. Add 500 mL Blocking Solution to wash the beads. This can be done by removing the rack from the magnet and inverting the rack with tubes still in place for 20 times or until the beads are evenly distributed in the Blocking Solution. c. Repeat the above wash twice, to reach a total of three washes. d. Resuspend the washed beads in 250 mL Blocking Solution, add 1 mg antibody, and put on a rotator at 4 C for at least 6 h or overnight (12-20 h).

Timing: 1 h hands-on time and overnight immunoprecipitation
This section describes the procedures to solubilize and break chromatin into appropriate size range, and use the specific antibody to immunoprecipitate the DNA bound by the protein of interest.
5. If using the frozen pellet from the steps described above, take the pellet from À80 C and thaw on ice. 6. Resuspend the pellet of appropriate cell numbers in 300 mL Sonication/IP Buffer with freshly added 13 protease inhibitor cocktails, and sonicate on a Bioruptor Pico (or the alternatives) for an appropriate number of cycles based on the results from Before You Begin section. 7. Incubate the chromatin with antibody-beads complex: a. Centrifuge the sonicated chromatin at 16,000 3 g at 4 C for 10 min. b. During the 10-min centrifugation time, wash the antibody-beads complex from step 4d three times with 500 mL Blocking Solution in the same way as described in steps 4a and 4b. c. Save 2 mL supernatant from step 7a, and store in À20 C as the input sample, and transfer the rest supernatant to the washed antibody-beads complex. Incubate overnight (12-20 h) at 4 C on a rotator.
Note: There should be very tiny or no visible pellet after the centrifugation at step 7a.

Wash Beads, Tagmentation on Beads, and Reverse Crosslinking
Timing: 1 h hands-on time and 6 h to overnight reverse crosslinking This section describes the procedures in to wash the immunoprecipitation and add sequencing adapters via tagmentation by Tn5. Then the crosslink is reversed by heating at 65 C. All wash steps are done at the bench with wash buffers kept on ice.
8. Put the immunoprecipitation on DynaMag-2 to collect the beads at the side of the tube. Invert twice or three times to collect beads at the tube cap. No need to centrifuge. 9. Remove the supernatant, and the buffer at the cap using an aspirator or a pipette. 10. Wash once with 500 mL RIPA Wash Buffer. This can be done by removing the tube rack from the magnet, add the buffer and invert by hand with the tube still on the rack for 15-20 times or until the beads are evenly distributed in the buffer. 11. Wash once with 500 mL Low Salt Wash Buffer. 12. Wash once with 500 mL High Salt Wash Buffer. 13. Wash once with 500 mL LiCl Wash buffer. 14. Wash twice with 500 mL 10 mM Tris-HCl, pH 8.0.

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Note: The washes from step 11 to 14 are performed in the same way as described in step 10.
CRITICAL: During the wash in step 14, the beads will not attach to the magnet very tightly. Therefore, DO NOT use the aspirator to remove the buffer. Instead, use a pipette to remove the buffer carefully.
15. Collect the beads to the bottom of the tube by a brief centrifugation at 100 3 g for 30 s. 16. Put the tube on DynaMag-2 and remove trace of Tris-HCl. 17. Resuspend the beads thoroughly with 30 mL tagmentation mix, which consists of 15 mL 23 TD Buffer + 14 mL ddH 2 O + 1 mL Tn5. The Tn5 can be from either the Illumina Tagment DNA TDE1 Enzyme and Buffer kit or the Fapon Tnp Library Prep Kit for Illumina. You only need one kit, not both. 18. Take the 2 mL input sample from À20 C, and mix with 30 mL tagmentation mix (the same as above). 19. Put both the IP and input samples on the thermomixer to incubate at 37 C for 5 min with 800 rpm shaking.
CRITICAL: This incubation step allows Tn5 to add sequencing adapters to the immunoprecipitated DNA. At this stage, the DNA is still bound by the protein which protects the DNA from being cut by Tn5. Therefore, you do not need to worry about over-tagmentation. We used 1 mL for easy pipetting regardless of the cell number used here. What happens in the tube is shown in Figure 5.

Stop the tagmentation reaction:
a. For input samples, directly add 70 mL ChIP Elution Buffer, add 1 mL of Proteinase K (20 mg/ mL) and leave at 65 C on a thermomixer with 1,400 rpm shaking for at least 6 h or overnight (12-20 h). b. For IP samples, wash beads with 500 mL Low Salt Wash Buffer twice, then with 500 mL 13 TE 50 mM NaCl once. Perform the wash in the same way as described in step 10. c. Briefly centrifuge the IP samples to collect beads at the bottom of the tube. Put the tubes to DynaMag-2 and remove trace of 13 TE 50 mM NaCl. d. Add 100 mL ChIP Elution Buffer to the beads, add 1 mL Proteinase K (20 mg/mL) and briefly vortex until the beads become homogeneous. e. Leave all samples at 65 C on a thermomixer with 1,400 rpm shaking for at least 6 h or overnight (12-20 h).
CRITICAL: In step 20c, the beads will not attach to the magnet very tightly. Therefore, DO NOT use the aspirator to remove the buffer. Instead, use a pipette to remove the buffer carefully. In step 20d, take care not to vortex the beads to the cap.

DNA Purification and Library Preparation
Timing: 2 h This section describes the procedures of DNA purification and library preparation. See Figure 6 below for the schematic view of this section with PCR details.
21. Purify DNA from both input and IP samples using the Qiagen minElute PCR Purification Kit according to manufacturer's instructions. Elute the DNA in 11 mL Elution Buffer from the kit twice, which generally yields 20 mL DNA.
Note: There is no need to quantify the DNA concentration at this stage. Use all for the next step.

OPEN ACCESS
STAR Protocols 1, 100187, December 18, 2020 22. Setup the library PCR reaction per sample as follows: 23. Run a pre-amplification PCR using the following condition: Note: The combination of S5xx and N7xx primers identifies a sample. Therefore, different samples should use different combinations of S5xx and N7xx primers. If you do not have many samples, it is recommended to use different N7xx primers, because the index in the N7xx primer is sequenced first on an Illumina machine.
CRITICAL: Since the tagmentation process creates 9-bp gaps (see Figure 5), the first step in the PCR should be 72 C to allow the polymerase to fill in the gaps.
24. After the pre-amplification, take out 9 mL of the reaction, and mix with 1 mL 103 EvaGreen and perform a qPCR analysis to decide the optimal cycle number. Leave the rest 41 mL reaction on ice. Note: The cycle number should be chosen at the exponential phase, before reaching saturation.
27. Once the optimal cycle number is decided, amplify the rest 41 mL reaction for a further of N cycles, using the following condition:  CRITICAL: Typically, the number N is between 6 and 14, i.e., the total number of cycles needed is between 10 and 18 cycles depending on the number of cells and the abundance of the protein. See Troubleshooting 2.
28. Purify the library PCR product using 1.23 beads ratio using AmpureXP for PCR Purification beads or VAHTS DNA Clean Beads, according to manufacturer's instructions. Elute the library using 30 mL Elution Buffer from the Qiagen minElute PCR Purification kit or 10 mM Tris-HCl, pH 8.0.

EXPECTED OUTCOMES
The quality and quantity of the purified library should be checked by an Agilent Bioanalyzer 2100 machine or the like. We use the Agilent High Sensitivity DNA Kit and follow exactly the steps described in the manufacturer's manual. Figure 8 shows a few examples of successful libraries and a failed one in different machines. One should expect at least 4 nM at the region between 200 bp and 1,000 bp.
Note: The shape of the size distribution of the library depends on many factors, such as the sonication and the protein of being analyzed. The majority of the DNA should fall between 200 and 1,000 bp. We found the large fragments (>1,000 bp) do not affect quantification or sequencing at all. Therefore, we just leave them as they are. Asterisks indicate primer leftover, which can be removed by a further beads purification if needed.
If the libraries look good, send for sequencing. We normally perform 50 bp pair end sequencing, but single end sequencing can also be used. We followed the ENCODE ChIP-seq guide (Landt et al., 2012) to sequence at least 20 million reads for each experiment, which is usually enough for point

QUANTIFICATION AND STATISTICAL ANALYSIS
Unfortunately, a successful library preparation does not mean a successful ChIPmentation experiment. Some preliminary computational analysis on the data need to be performed to see if the ChIPmentation experiment is working or not.

Sequence Read Alignment
Timing: 10 min to hours depending on computing power and sequencing depth Use the following command for the read alignment and format conversion: The commands align the reads to the genome, remove un-aligned reads, sort the reads by coordinates and only keep reads with mapping quality higher than 30.

Peak Calling
Timing: at least 10 min depending on sequencing depth Check the MACS2 manual for more information.
There will be a file in bedGraph format called {factor}_treat_pileup.bdg generated after the MACS2 peak calling. It is recommended to convert it to the bigWig format for the visualization. First, get the chromosome sizes for the genome, using the human genome hg38 as an example:

Assessment of Results
It is difficult to define universal rules to check whether a ChIPmentation experiment works or not. In our experience, the peak file generated during the peak calling process by MACS2 should contain thousands or even tens of thousands of peaks when an experiment is successful. However, some factors may have very few binding peaks. We have found visual inspection of the binding signal in the bigWig file using UCSC genome browser can be very helpful. See Troubleshooting 3.
First, look at the chromosome-wide view of the experiment. For successful experiments, clear ''spikes'' should be apparently visible to eyes, and there should be many comparing to the input sample. For failed experiments, it is relatively flat. When zooming in into specific target genes, you should see smooth bell-curve shaped peaks. The peak and the background can be easily discriminated by eyes. See examples in Figure 9.

LIMITATIONS
The nature of ChIPmentation is essentially ChIP-seq. Many limitations that restrict the use of ChIPseq also apply to ChIPmentation. The technique still requires a ChIP-grade antibody that can recognize and pull down its target protein after formaldehyde crosslinking. In general, finding a ChIPgrade antibody is difficult and time consuming. Alternatively, stable cell line expressing an epitope tagged version of the protein of interest can be generated, and an antibody against the tag can be used for ChIPmentation. For example, the ENCODE project has used the 3xFLAG tag to investigate genomic locations of hundreds of chromatin-associated proteins (Partridge et al., 2020), but this cannot be achieved in primary cells and tissues.
In addition, ChIPmentation only simplifies the procedure and increase the sensitivity of the library preparation steps. It does not change the chromatin immunoprecipitation part of the protocol. Therefore, the cell number required for a ChIPmentation experiment is relatively low comparing to the traditional ChIP-seq methods. In our hands, the minimum cell number for a successful ChIPmentation experiment is 10 4 for profiling histone modifications and 10 5 for investigating transcription factors. However, these numbers are still high and prohibit the profiling of rare material. If cell number is very limited, other methods such as uliChIP-seq (

TROUBLESHOOTING Problem 1
Poor sonication results: the majority of fragments are too large (>1 kb), or too small (100-200 bp) or heterogeneous (the presence of both large and small fragments at the same time), or not enough input DNA to visualize on agarose gels due to low number of cells.

Potential Solution
When either large fragments or small fragments are present, adjust the sonication condition accordingly. The most straightforward approach is to change the number of cycles of the sonication, but sometimes, one needs to change the ON/OFF time. For histone modification, small fragments may not be a big problem.
We have found heterogeneous sonication often results from heterogeneous crosslinking. This often happens for cells that grow in colonies (i.e., not monolayer) or for primary cells not properly dissociated from tissues. Optimize your system to get good single-cell suspension first (i.e., use trypsin, collagenase etc.), and start from step 1 of the Step-By-Step Method Details section to crosslink the cells in solution.
If cells number is limited, and not enough DNA is recovered, one can reverse crosslink and purify DNA from the entire sample (instead of taking a fraction out).

Problem 2
Low yield of immunoprecipitated DNA: this can be reflected at the qPCR stage where N (is step 26) is high (>14) or a flat profile on Bioanalyzer or the like.

Potential Solution
It should be noted that high number of cycles does not necessarily mean a failed experiment, but a flat profile indicates the experiment probably failed. This could be due to low abundance of the protein of interest, or low antibody affinity to the protein. Increase the starting number of cells to see if it helps. In addition, trying different antibodies or adding an epitope tag to the protein of interest often help. Commonly-used tags with good antibodies include 3xFLAG, V5, and 3xHA. See the ENCODE ChIP-seq guide (Landt et al., 2012) for more details.

Problem 3
Library preparation is successful, but the sequencing results suggest low signal-to-noise ratio. This is the most frequently encountered problem according to our experience.

Potential Solution
Like suggested in the previous section, a successful library does not necessarily mean a successful ChIPmentation. Include a positive control antibody, such as a transcription factor antibody that has been tested successfully in ChIP-seq/ChIPmentation by neighboring labs to make sure the protocol is working as intended. If the positive control works, but the actual experiment fails, try to change to a different antibody or considering adding tags to the protein of interest.
Another reason could be the protein of interest does not interact with DNA tightly or directly, a dual crosslinking step can be used. Formaldehyde can crosslink both protein-DNA and protein-protein interactions, but it has poor efficiency of crosslinking protein-protein interactions due to its short spacer arm. A protein-protein crosslinker with a longer spacer arm (such as EGS) can be used to secure protein-protein interaction first, then formaldehyde is used to crosslink protein-DNA interaction. Check (Zeng et al., 2006) for details.
Finally, the right crosslinking condition also needs to be tested. If under-crosslinking happens, the protein will not be efficiently crosslinked to DNA and the bound DNA may be lost during the washes. If over-crosslinking happens, the protein epitope will be destroyed and the antibody will not be able to recognize the protein of interest. Both cases result in low signal-to-noise ratio. We suggest choosing a time course of crosslinking and perform the ChIPmentation experiment in parallel to find the condition that gives the best signal-to-noise ratio.

Lead Contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Xi Chen (chenx9@sustech.edu.cn).

Materials Availability
This study did not generate new unique reagents. All materials used in this study are commercially available, and the detailed information can be found in the Key Resources Table. Data and Code Availability This study did not generate new data or code.