- 1 Introduction
- 2 Materials
- 3 Methods
Chromatin is a complex biomolecule. It is dynamic in nature and it is responsive to intra and extra cellular signal regulating DNA metabolism. Chromatin plays a key role in every DNA metabolism like transcription, DNA repair, DNA replication and chromosome condensation. Chromatin Immunoprecipitation (ChIP) is the technique used to study the interaction of proteins and DNA molecules. The technique is also used for estimation of the density of the interaction.
The ChIP assay represents a major advancement in the study of chromatin processes and its use has increased dramatically over the last few years. The ChIP assay begins with the cross-linking of protein–DNA complexes by the fixation of cells/tissues with formaldehyde. After lysing the cells, the nuclei are disrupted and the chromatin is sheared either by sonication or by digestion with micrococcal nuclease. The chromatin fragments, typically between 500 and 1,000 base pairs in length, are immunoprecipitated using an antibody specific to the protein of interest. After reversing the cross-links, the DNA is isolated and used in one of the several detection methods including dot/slot blot, PCR or qPCR, hybridization to a DNA microarray (ChIP-chip), or sequenced using a rapid sequencing technology (ChIP-seq). Enrichment of a particular DNA region over other sites where the factor is not expected to bind indicates that the protein interacts with this region.
The traditional ChIP assay, though it has proved to be powerful, is time-consuming and laborious. The slowest step of the traditional ChIP assay is the 5 hour reversal of cross-linking and the most laborious step is the DNA cleanup, which involves phenol: chloroform extraction and ethanol precipitation. In Fast ChIP, cross-links are reversed during a 10 min incubation at 100 C in the presence of Chelex-100. In addition, since Fast ChIP does not require the addition of sodium bicarbonate/SDS buffer to elute the chromatin from the beads, the DNA cleanup step is not necessary. After the 10 C incubation, the DNA-containing supernatant is directly used in PCR. Thus, several hours and a great deal of labor in the traditional assay are replaced with a 10 min incubation in Fast ChIP.
Another improvement in Fast ChIP is the use of an ultrasonic bath to increase the rate of antibody–chromatin interaction. In the traditional ChIP assay, the antibody–chromatin incubation can take anywhere from 1 h to overnight. With the use of the ultrasonic bath, this incubation is decreased to 15 min. The combination of these two improvements in Fast ChIP not only allows the assay to be easily completed in 1 day, starting with sonicated chromatin extracts but also gives enough time for the products to be analyzed by qPCR in the same day.
Due to its simplicity and reduced labor, Fast ChIP facilitates studies which involve multiple chromatin samples, multiple antibodies, or both. These include studies where (i) multiple proteins or protein modifications (e.g., histone modifications) are observed simultaneously; (ii) multiple time points are observed; or (iii) antibodies and chromatin extracts are being screened for their suitability in ChIP. Beginning with sheared chromatin, 24 ChIP samples can be easily processed to yield PCR-ready DNA in 5 h. Also, the short time required for completion of the assay is helpful when optimizing conditions for a particular antibody or when learning the assay for the first time. We have used Fast ChIP with chromatin from tissue culture, mammalian tissues, and yeast cultures, and it is likely that it is compatible with most other sources of chromatin. Though we have designed Fast ChIP for analysis by PCR or qPCR, it has also been used, with the addition of a column cleanup step, in ChIP-chip studies. Thus, it is likely that Fast ChIP may be used for most ChIP applications, including ChIP-seq.
- Protein A–Sepharose
- Phosphate buffered saline (PBS).
- SYBR Green PCR Master Mix.
Buffers and Solutions
- 1 M Glycine.
- IP buffer: 150 mM NaCl, 50 mM Tris–HCl, pH 7.8, 5 mM EDTA, pH 8.0, 0.5% (v/v) NP-40, 1% (v/v) Triton X-100.
- Lysis/sonication buffer: make it fresh before each use. Per 1 mL of IP buffer, add the following protease inhibitors: 5 mL PMSF (0.1 M in isopropanol; stored at –20 deg._C; re-dissolve at room temperature before pipetting) and 1 mL leupeptin (10 mg/mL; aliquoted and stored at –20_deg. C) and keep on ice. In addition, the following phosphatase inhibitors may be added if required for ChIP with phosphospecific antibodies: 10 mL b-glycerophosphate (1 M; stored at 4_deg. C), 10 mL sodium fluoride (1 M; stored at 4_deg. C; resuspend before pipetting), 10 mL sodium molybdate dihydrate (10 mM; stored at 4_deg. C), 1 mL sodium orthovanadate (100 mM; stored at –20_deg. C), and 13.84 mg p-nitrophenylphosphate (stored at 4_deg. C).
- 10% Chelex-100 in ddH2O.
- 20 mg/mL proteinase K in ddH2O.
- TE, pH 9.0: 10mMTris–HCl, 1mMEDTA, bring to pH9.0 with 5 M NaOH.
- Sonicator with microtip (e.g., Misonix Sonicator 3000).
- Refrigerated microcentrifuge.
- Heat blocks and hot plate (for 55_deg. C incubation and boiling water incubation).
- Tube rotator or tumbler at 4_deg. C.
- Set-up for quantitative PCR.
- Ultrasonic bath (optional).
The steps which make Fast ChIP unique compared to other ChIP methods are immunoprecipitation and preparation of PCR-ready DNA. Therefore, the following methods for cross-linking, lysis, and sonication are based on what has worked but are certainly not the only methods compatible with Fast ChIP. If a researcher has previously established his/her own chromatin preparation method for ChIP, they should continue to use this method with Fast ChIP. To ensure equal loading of different chromatin samples, especially necessary when tissue fragments are used, we suggest extracting total DNA from each chromatin sample and measuring the amount of DNA for each by qPCR. If the samples differ by more than 25%, the amount of chromatin loaded should be adjusted based on this measurement. If the amount of chromatin is adjusted, remember to use an average of the input samples while calculating the percent of input. If extracting the input DNA for quantitation to adjust chromatin loading for ChIP (especially if using tissue samples) or for analyzing the chromatin fragmentation (optimizing the sonication conditions), we suggest doing the cross-linking, lysis, and sonication steps on a separate day from the ChIP. If using cells from tissue culture, equal chromatin loading can be more easily controlled than in tissue samples by ensuring equal density on plates. Therefore, if sonication conditions have already been optimized, for tissue culture the entire assay can be completed in 1 day with the input DNA extraction and the ChIP being processed simultaneously.
- Keep in mind that approximately 4*105–106 cells are required per IP sample.
- Add 40 mL 37% formaldehyde per milliliter of tissue culture medium directly to the dish/flask (1.42% final concentration), swirl, and incubate at room temperature for 15 min.
- Quench formaldehyde by adding 141 mL of 1M glycine per milliliter of medium (125 mM final concentration) and incubate for 5 min at room temperature.
- Harvest cells by scraping and centrifuging at 2,000g for 5 min (4_deg. C).
- Keep cells on ice and wash twice with ice-cold IP buffer. After aspirating the PBS, the cell pellet can be stored at –80_deg. C for at least a year.
Fresh or Frozen Tissue
This method has been used in our laboratory for ChIP on both kidney and liver tissue and is likely to be effective in other tissues which have similar numbers of cells per volume of tissue.
- Place approximately 0.1 cm3 piece of fresh or frozen (–80 deg._C) tissue in 1 mL of PBS containing 1% formaldehyde at room temperature and quickly mince with forceps into 1–2 mm3 fragments.
- Incubate tissue fragments at room temperature for 20 min.
- Centrifuge at 2,000–3,000g for 1 min (4_deg. C) and discard the supernatant.
- Suspend pellet in 1 mL PBS with 125 mM glycine and incubate for 5 min at room temperature.
- Centrifuge tissue fragments, and discard the supernatant.
- Wash twice with PBS and place on ice for the lysis/sonication step.
For both cross-linking and lysis of yeast cells, we use the method described by Kuo and Allis up to the point where the whole cell lysate is obtained. At this point, Fast ChIP can be used, beginning at the sonication steps.
- Lyse approximately 107 cells by resuspending in 1 mL ice-cold lysis/sonication buffer and pipetting up and down several times.
- Collect the insoluble material, which includes the nuclei, by centrifuging at 12,000g for 1 min (4_deg. C), and aspirate the supernatant.
- Resuspend the pellet once more in 1 mL lysis/sonication buffer, collect the pellet by centrifugation, and aspirate the supernatant. This washes away residual soluble proteins from the pellet leaving insoluble chromatin, nuclear matrix, and the associated cytoskeleton.
- For tissues, resuspend cross-linked fragments in 1 mL lysis buffer and proceed to the sonication step.
- Resuspend the pellet in 1 mL ice-cold lysis/sonication buffer, and split into two 500 mL fractions. At this point, both the fractions should be in 1.5 mL microcentrifuge tubes. Both the volume of buffer and the geometry of the tube used for sonication affect fragmentation efficiency with volumes of 500 mL or less and 1.5 mL microcentrifuge tubes (for tissues 1 mL buffer and 2 mL tubes) being optimal.
- The protocol used for sonication can vary widely and must be optimized for each cell or tissue type and sonicator setup. Optimal fragment sizes are typically between 0.5 and 1 kb as determined by running sonicated chromatin on 1% agarose after DNA extraction and reversal of cross-links. The following are suggestions for optimizing sonication using a microtip:
- Sonication can cause heating of the sample; so the tube should be immersed in an ice-water bath during sonication.
- Foaming can occur if the microtip gets too close to the surface of the sample during sonication. The tip should remain no more than a few millimeters from the bottom of the tube during sonication. If foaming does occur, stop sonication and wait till the majority of bubbles rise to the surface before continuing sonication.
- The two variables to optimize are the total amount of sonication time and the power output of the sonicator.
- To avoid excessive heating, the total sonication time should be broken up into rounds of 10–20 s each, with at least 2 min of rest on ice between each round. In addition, sonication is more efficient if each round is broken up into approximately 1 s pulses rather than continuous sonication since the power of sonication decreases gradually after the beginning of each pulse.
- The higher the power output of the sonicator the faster the fragmentation of the chromatin and the more heating the sample is exposed to. Start with a power output 50% or less of the total power output for the sonicator and increase as needed such that the samples are not overheated by the end of each round of sonication, but the amount of time required for sonication is not prohibitive considering the number of samples to be sonicated.
- Other factors which affect sonication efficiency are the cell concentration and the extent of cross-linking of the chromatin. Diluting the chromatin and/or reducing the cross-linking time or concentration of formaldehyde can increase sonication efficiency.
- After sonication, the chromatin should be cleared by centrifugation at 12,000g for 10 min (4 C).
- Transfer the supernatant to a new tube and aliquot for storage at –80 C. Save one aliquot of 10 mL for extracting total DNA for the ‘input’ sample.
Isolating Total DNA (Input Sample)
Unless otherwise stated, Steps 1–14 can be performed at room temperature.
- Precipitate DNA from the 10 ml aliquot from sonication Step4 for 10 min at room temperature with 30 mL absolute or 96% ethanol.
- Pellet the DNA by centrifugation at 12,000g for 3 min (4 C).
- Aspirate or decant the supernatant and add 50 mL 75% ethanol.
- Centrifuge at 12,000g for 1 min (4 C), and remove as much of the supernatant as possible.
- Dry the pellets to completion (they should become transparent after drying).
- Add 100 mL of 10% Chelex-100 slurry to the dried pellets.
- Boil for 10 min and cool by centrifuging for 1 min (4 C).
- Add 1 mL of 20mg/mL proteinase K to each tube and vortex. Briefly, centrifuge to bring contents to the bottom of the tube.
- Incubate at 55 C for 30 min, gently resuspending the Chelex once or twice during the incubation.
- Boil for 10 min and centrifuge the condensate to the bottom of the tube at 10,000g for 1 min (4 C).
- Transfer 80 mL of the supernatant to a new tube.
- Add 120 mL ddH2O to each tube containing Chelex slurry, vortex, and centrifuge the contents to the bottom of the tube.
- Remove 120 mL of the supernatant and pool with the 80 mL supernatant from Step 10.
- The DNA can be run undiluted on 1% agarose. For PCR, use no less than a 1:20 dilution in TE, since some of the remaining contaminants can be inhibitory to PCR.
- For each IP sample, dilute the equivalent of 1*106 cells of chromatin to 200mL with ice-cold lysis/sonication buffer.
- Add specific or mock antibodies to each sample and mix by inverting.
- Turn the ultrasonic bath on and float samples in the bath for 15 min at 4 C.
- Clear the solution by centrifugation at 12,000g for 10 min (4 C). This step is essential to remove non-specific insoluble chromatin aggregates which may contaminate the final product.
- While the chromatin and antibodies are incubating, transfer approximately 20 mL per IP sample of proteinA agarose slurry to a clean tube. Wash 1–3 times with IP buffer to remove ethanol.
- Resuspend beads in 180mL IP buffer for every 20mL of beads. Dispense 200 mL of the diluted slurry to new tubes, 1 tube for each IP sample. Centrifuge and aspirate buffer. Visually inspect tubes to make sure each one has the same amount of beads.
- Transfer no more than the top 90% of each cleared chromatin sample from Step 4 (avoiding the pellet at the bottom of the tube) to the tubes with the beads.
- Rotate tubes at 4 C for 45 min with a rotating platform or tumbler. The rotation should be fast enough to keep the beads suspended.
- Centrifuge the tubes at 10,000g for 1 min (4 C) and aspirate the supernatant.
- Wash the beads (resuspend with buffer, centrifuge, and aspirate the supernatant) five times with 1 mL ice-cold IP buffer. After the last wash, remove as much supernatant as possible without removing the beads.
- Add 100 mL of 10% Chelex-100 slurry to the washed beads.
- Add 1 mL of 20 mg/ mL proteinase K to each tube and vortex. Briefly, centrifuge contents to the bottom of the tube.
- Incubate at 55_deg. C for 30 min. Gently resuspend beads and Chelex-100 once or twice during the incubation.
- Boil samples for 10 min.
- Centrifuge samples at 10,000g for 1 min (4 C) to cool samples and bring condensate to the bottom of the tube.
- Transfer 80 mL of supernatant to new tubes.
- Add 120 mL ddH2O to each tube containing Chelex/protein, beads slurry, vortex, and centrifuge contents to the bottom of the tube.
- Remove 120 mL of the supernatant and pool with the 80 mL supernatant from Step 16.
- The PCR-ready DNA can be stored at –20 C and repeatedly thawed and frozen over several months without loss of PCR signal.
PCR and Calculation of Enrichment
We use 2.35 mL of IP DNA or diluted input DNA in 5 mL reactions with 0.15 mL of the primer pair (each primer at 10mM), and 2.5 mL of master mix (SensiMix containing SYBR green and ROX) in 384-well PCR plates. The reactions are run in triplicate in 384-well PCR plates on the ABI 7900 for 40 cycles with the default two-step method. Data are acquired and analyzed using the SDS software. The threshold is set manually and Cts are imported to EXCEL for calculations.
We express enrichment of the immunoprecipitated region of the genome as the percent of input DNA. To eliminate the differences in amplification efficiencies of different primers, relative amounts of DNA for the IP, mock, and input samples are calculated for each primer using a standard curve. The standard curve consists of serial dilutions of total DNA from the same cell type or tissue used in the experiment and is run each time a primer pair is used. We suggest making up a large amount of each dilution in TE buffer and aliquoting them for multiple uses so that the standard curve can be run repeatedly without error due to degradation of the DNA.
PCR-primer efficiency curves are fit to the natural log of concentration vs. Ct for each dilution using an r-squared best fit. The relative amount for each ChIP and input DNA sample is calculated from their respective averaged Ct values using the formula:
where b and m are the curve fit parameters from the primer calibration curve that is generated for each PCR experiment. Dilution is the cumulative dilution of ChIP DNA as compared to the input DNA sample. Final results are expressed either as a fraction or percent of input using the following equation:
% of input=([DNA sample] – [DNA mock]/ [DNA input]) * 100
where DNA concentrations were computed from equation . DNA sample is the ChIP DNA sample, DNA mock is the IgG mock IP control, and DNA input is the input DNA used in ChIP. Remember that, if the chromatin amount used in ChIP was adjusted based on measurement of the input samples, then DNA input in equation  should be an average of the input for all the samples.
The enrichment (percent of input) determined using the above calculations is, in itself, not a meaningful number. To determine the significance of the enrichment at a region of interest, this region must be compared to another region where the factor of interest is not expected to bind (the negative control region). The enrichment at the negative control region gives a baseline which is assumed to represent zero binding and the significance of the enrichment of the region of interest depends on the signal at this region being significantly above the baseline. Another means of determining the significance of enrichment of a factor at a particular locus is to compare that enrichment in cells where the factor is present to those where the factor has been knocked out. The enrichment in the knockout cells represents the zero binding baselines, and enrichment is significant at the region of interest only if it is above this baseline.