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UNIT 17.14 Chromatin Immunoprecipitation of Adult Murine Cardiomyocytes P. Bolli, 1 C. Vardabasso, 2 E. Bernstein, 2 and H.W. Chaudhry 1 1 Department of Cardiology, Mount Sinai School of Medicine, New York, New York 2 Departments of Oncological Sciences and Dermatology, Mount Sinai School of Medicine, New York, New York ABSTRACT This unit describes a streamlined two-step protocol for the isolation of adult murine cardiomyocytes with subsequent Chromatin ImmunoPrecipitation (ChIP). Isolation and culturing of cardiomyocytes is a delicate process and the protocol presented here op- timizes the combination of cardiomyocyte isolation with ChIP. ChIP is an invaluable method for analyzing molecular interactions occurring between a specific protein (or its post-translationally modified form) and a region of genomic DNA. ChIP has be- come a widely used technique in the last decade since several groundbreaking studies have focused attention on epigenetics and have identified many epigenetic regulatory mechanisms. However, epigenetics within cardiovascular biology is a new area of focus for many investigators, and we have optimized a method for performing ChIP in adult murine cardiomyocytes, as we feel this will be an important aid to both the cardiovascular field and for the development of cell- and tissue-specific ChIP. Curr. Protoc. Cell Biol. 58:17.14.1-17.14.16. C 2013 by John Wiley & Sons, Inc. Keywords: Chromatin ImmunoPrecipitation adult murine cardiomyocytes cardiomyocyte isolation BASIC PROTOCOL Only recently have the epigenetic regulators of key cardiac developmental genes begun to be discovered, and a growing body of evidence has now highlighted the effects of chromatin organization on the activity of the cardiac transcriptional network (Sdek et al., 2011; He et al., 2012; Lee et al., 2012; Wamstad et al., 2012). Chromatin ImmunoPrecipitation (ChIP) is an invaluable method for analyzing molecular interactions occurring between a specific protein (or its post-translationally modified form) and a region of genomic DNA. When performing ChIP, cells are first fixed with formaldehyde to covalently cross-link proteins to DNA. Then, chromatin is isolated from the cells, sheared, and subjected to an immunoselection process, which requires the use of specific antibodies. Any DNA sequence cross-linked to the protein of interest will coprecipitate as part of the chromatin complex. After the immunoselection of chromatin fragments and purification of associated DNA, the detection of specific DNA sequences is performed by quantitative PCR (qPCR) or by standard PCR followed by gel elec- trophoretic analysis. Any DNA sequence specifically associated with the protein being examined will be increased (or enriched) by the immunoprecipitation process, and this will be detected by qPCR. ChIP can be difficult when studying cardiomyocytes as it requires large cell numbers (10 6 -10 7 ). Adult mouse cardiomyocyte isolation is challenging and often does not provide the required number of cells. In this unit we present a detailed protocol for cardiomyocyte isolation followed by ChIP obtained by modification of standard protocols. The present Current Protocols in Cell Biology 17.14.1-17.14.16, March 2013 Published online March 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/0471143030.cb1714s58 Copyright C 2013 John Wiley & Sons, Inc. Macromolecular Interactions in Cells 17.14.1 Supplement 58

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Page 1: Current Protocols in Cell Biology || Chromatin Immunoprecipitation of Adult Murine Cardiomyocytes

UNIT 17.14Chromatin Immunoprecipitation of AdultMurine Cardiomyocytes

P. Bolli,1 C. Vardabasso,2 E. Bernstein,2 and H.W. Chaudhry1

1Department of Cardiology, Mount Sinai School of Medicine, New York, New York2Departments of Oncological Sciences and Dermatology, Mount Sinai School of Medicine,New York, New York

ABSTRACT

This unit describes a streamlined two-step protocol for the isolation of adult murinecardiomyocytes with subsequent Chromatin ImmunoPrecipitation (ChIP). Isolation andculturing of cardiomyocytes is a delicate process and the protocol presented here op-timizes the combination of cardiomyocyte isolation with ChIP. ChIP is an invaluablemethod for analyzing molecular interactions occurring between a specific protein (orits post-translationally modified form) and a region of genomic DNA. ChIP has be-come a widely used technique in the last decade since several groundbreaking studieshave focused attention on epigenetics and have identified many epigenetic regulatorymechanisms. However, epigenetics within cardiovascular biology is a new area of focusfor many investigators, and we have optimized a method for performing ChIP in adultmurine cardiomyocytes, as we feel this will be an important aid to both the cardiovascularfield and for the development of cell- and tissue-specific ChIP. Curr. Protoc. Cell Biol.58:17.14.1-17.14.16. C© 2013 by John Wiley & Sons, Inc.

Keywords: Chromatin ImmunoPrecipitation � adult murine cardiomyocytes �

cardiomyocyte isolation

BASICPROTOCOL

Only recently have the epigenetic regulators of key cardiac developmental genes begunto be discovered, and a growing body of evidence has now highlighted the effects ofchromatin organization on the activity of the cardiac transcriptional network (Sdek et al.,2011; He et al., 2012; Lee et al., 2012; Wamstad et al., 2012).

Chromatin ImmunoPrecipitation (ChIP) is an invaluable method for analyzing molecularinteractions occurring between a specific protein (or its post-translationally modifiedform) and a region of genomic DNA. When performing ChIP, cells are first fixed withformaldehyde to covalently cross-link proteins to DNA. Then, chromatin is isolated fromthe cells, sheared, and subjected to an immunoselection process, which requires the useof specific antibodies. Any DNA sequence cross-linked to the protein of interest willcoprecipitate as part of the chromatin complex. After the immunoselection of chromatinfragments and purification of associated DNA, the detection of specific DNA sequencesis performed by quantitative PCR (qPCR) or by standard PCR followed by gel elec-trophoretic analysis. Any DNA sequence specifically associated with the protein beingexamined will be increased (or enriched) by the immunoprecipitation process, and thiswill be detected by qPCR.

ChIP can be difficult when studying cardiomyocytes as it requires large cell numbers(106-107). Adult mouse cardiomyocyte isolation is challenging and often does not providethe required number of cells. In this unit we present a detailed protocol for cardiomyocyteisolation followed by ChIP obtained by modification of standard protocols. The present

Current Protocols in Cell Biology 17.14.1-17.14.16, March 2013Published online March 2013 in Wiley Online Library (wileyonlinelibrary.com).DOI: 10.1002/0471143030.cb1714s58Copyright C© 2013 John Wiley & Sons, Inc.

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cross-linking the cardiomyocytes

isolation of DNA by cellular and nuclear lysis

sonication

immunoprecipitation with antibody of interest

magnetic separation

DNA purification

real-time PCR

cardiomyocyte isolation

Figure 17.14.1 Schematic of the protocol.

protocol allows optimization of the cell number, buffers, and timing (Fig. 17.14.1).Moreover, we provide recipes to make homemade buffers thus reducing costs.

Materials

Perfusion buffer (see recipe)Digestion buffer (see recipe)Stop buffer 1 (see recipe)Stop buffer 2 (see recipe)Cardiomyocyte medium (see recipe)IsofluraneAdult mouse (the type of mouse used will depend on the aim of the experiment)70% ethanolCollagenase D (Roche)2,3 butanedione monoxime (BDM; Sigma)Calcium chloride (CaCl2)Liquid nitrogen

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IcePhosphate-buffered saline (PBS; Mediatech, cat. no. 21-030)Protease inhibitor cocktail (Roche, cat. no. 118361770001)16% formaldehyde solution (Electron Microscopy Sciences) or 18.5%

formaldehyde solution (see recipe)Glycine (see recipe)Cell lysis buffer (see recipe)Nuclei lysis buffer (see recipe)RNase, optionalProteinase K, optionalAgarose gel, optionalGel loading buffer, optionalGel loading dye, optional100-bp DNA marker, optionalDilution buffer (see recipe)Antibodies to protein of interest (preferentially ChIP-grade)Antibody (Normal rabbit IgG)Protein A magnetic beads (alternatively Protein G magnetic beads)Low salt wash buffer (see recipe)High salt wash buffer (see recipe)LiCl wash buffer (see recipe)TE buffer (see recipe)Elution buffer (see recipe)Molecular grade water (Mediatech, cat. no. 46-000)Primers specific for analysis of the regions of interest2× Sybr green (Roche, cat. no. 04673484001)

37◦C water bathPerfusion apparatus: Langendorff apparatusSurgical apparatus (e.g., prepare a surgical table by wrapping a polystyrene lid with

aluminum foil)Gel loading tipsBlade22-G feeding needle, straight, with a ball tip, optional100-mm petri dishesBraided silk suture (6-0, metric 0.7)Dissecting microscope1-ml syringesSurgical tableSurgical instruments (all surgical tools are from Fine Science Tools) including:

Dumont #5 fine-tip forceps for the aorta cannulationSmall scissors

Laminar flow culture hood1000-μl pipet tips100-μm filters15- and 50-ml conical tubesCentrifugeHemacytometerIce bucketRotating platform, at 4◦C and at room temperatureVortex mixerSonicator (e.g., Bioruptor from Diagenode)1.5-ml microcentrifuge tubesHeating block preset to 95◦C

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Shacking incubator, preset at 62◦CGel electrophoresis apparatus, optionalMagnetic separation rackDNA purification kit (e.g., Qiagen)Thermal cycler

Additional reagents and equipment for animal euthanasia (Donovan and Brown,2006)

CAUTION: Formaldehyde is a biohazard. Consult MSDS for proper handling instruction.

NOTE: Any protocol using vertebrate animals must be approved by an InstitutionalAnimal Care and Use Committee (IACUC) and must follow approved procedures for thecare and use of laboratory animals.

Cardiomyocyte isolation (1 hr for preparation + 1 hr for each heart isolation)The cardiomyocyte isolation protocol is adapted from “Isolation and Culture of AdultMouse Cardiac Myocytes” by O’Connell et al. (2007).

1. Prepare 1× fresh perfusion buffer, digestion buffer without collagenase, stop buffers,and cardiomyocyte medium as described in the Reagents and Solutions section. Warmthe perfusion buffer, myocyte digestion buffer, and cardiomyocyte medium to 37◦Cin a water bath.

2. Prepare the perfusion and the surgical apparatus. Prepare the cannula by gentlyscoring a gel loading tip with a blade to create grooves. (Alternatively, a 22-Gfeeding needle, straight, with a ball tip can be used.) These will prevent slipping ofthe heart once it is cannulated.

3. Tape the cannula on the lid of a 100-mm petri dish, pass two 6-0 silk suture with a half-knot around the cannula and place it under the dissecting microscope (Fig. 17.14.2).Fill up a 1-ml syringe with the perfusion buffer. Run perfusion buffer through theperfusion system, avoiding bubbles.

4. Anesthetize the mouse with isoflurane and sacrifice by cervical dislocation (seeDonovan and Brown, 2006).

surgical instruments

syringe with perfusion buffer

cannula with silk suture

Figure 17.14.2 Cannulation setup.

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Figure 17.14.3 Diagrammatic illustration of the different steps to be performed during heart perfusion.

5. Transfer the mouse to the surgical table. Clean the chest with 70% ethanol. Openthe peritoneal cavity and chest with small scissors, peel back the rib cage, and cut toexpose the heart. Lift the heart and cut all the vessels (Fig. 17.14.3).

6. Transfer the heart into a 100-mm petri dish containing warm perfusion buffer andsqueeze it gently to remove all the blood.

7. Place the heart under the dissecting microscope (Fig. 17.14.3). Insert the cannula inthe aorta using fine-tip forceps to slide the aorta onto the cannula. Make sure that thetip of the aorta is just above the aortic valve (not into the ventricle). Tie the threadsaround the aorta (Fig. 17.14.3).

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8. Connect the syringe to the cannula and inject the heart with the buffer. If the cannulais placed correctly, the heart should become swollen.

9. Transfer the heart to the Langendorff apparatus.

10. Once cannulated, perfuse the heart with the perfusion buffer for 3 min. The heartshould start turning pale (Fig. 17.14.3).

11. Meanwhile add 40 mg collagenase D to the tube containing 10 ml digestion buffer.

Amount of collagenase needed may vary from different batches and for older enzyme.

12. Start perfusion of the heart with the digestion buffer and digest for 8 to 13 min (timeof digestion may vary from different batches and for older enzyme) at 4 ml/min.

After 2 min the digestion buffer can be collected and reused for the digestion.

If the heart is well perfused, it will become swollen and turn pale.

13. Once enzymatic digestion of the heart is complete (heart appears swollen, pale andflaccid), cut the heart from the cannula just below the atria. Place the ventriclesin a 100-mm petri dish (Fig. 17.14.3) and fill it with the digestion buffer from theperfusion apparatus (roughly 10 ml).

Move under a laminar flow culture hood.

14. Gently tease the ventricles into several small pieces with fine forceps (Fig. 17.14.3).Cut a 1000-μl pipet tip and pipet very gently (avoid bubbles) several times(Fig. 17.14.3); this process takes a few minutes.

15. Transfer the cells into a 15-ml conical tube with a 100-μm filter on the top.

16. Add 10 ml stop buffer A to the petri dish and rinse it well to collect all the cells.

17. Transfer the solution into the 15-ml conical tube with a 100-μm filter on the top tocombine with the cell suspension.

18. Centrifuge the tube for 2 min at 60 × g, room temperature.

19. Remove the supernatant and add 10 ml stop buffer B. Pipet very slowly and transferto a fresh petri dish.

20. Add 0.3 ml BDM.

21. Start a gradual calcium reintroduction as follows:

a. Add 50 μl of 10 mM CaCl2.

b. Mix by moving the dish forward and backward and side-to-side.

c. Incubate for 4 min at room temperature.

22. Add an additional 50 μl of 10 mM CaCl2. Mix and incubate for 4 min at roomtemperature.

23. Add 100 μl of 10 mM CaCl2. Mix and incubate for 4 min at room temperature.

24. Add 30 μl of 100 mM CaCl2. Mix and incubate for 4 min at room temperature.

25. Add 50 μl of 100 mM CaCl2. Mix well and incubate for 4 min at room temperature.

26. Transfer (pipetting slowly and washing the plate) the cells into a new 15-ml tube andcentrifuge for 2 min at 60 × g, room temperature.

27. Remove the supernatant and resuspend the cell pellet in 1 ml cardiomyocyte medium.Count the cells with a hemacytometer.

A good isolation should achieve a million rod-shaped cardiomyocytes.

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28. Centrifuge for 2 min at 60 × g, room temperature, and remove the supernatant. Thecell pellet can now be processed immediately or snap-frozen with liquid nitrogenand stored up to several months at −80◦C.

A plating step on a laminin-coated plate could be performed at this point to get rid ofdead cells (dead cells will not attach and can be washed away).

To prepare laminin-coated plates: The stock solution is 1 mg/ml, and should be stored in50-μl aliquots at −80◦C. The day before perfusion thaw the laminin in the refrigeratorand cover each 10-cm plate with 10 ml PBS + 50 μl laminin. Store the plate overnight inthe incubator. Non-used plates can be stored for several days at 4◦C.

Cardiomyocytes cross-linking (1 hr)Preparation: Obtain ice for incubation. Place 1× PBS on ice. Remove protease inhibitorcocktail and thaw it at room temperature.

29. Resuspend the cells in 10 ml cardiomyocyte medium (if using frozen cells add 10ml PBS to frozen cell pellet).

If working with adherent cardiomyocytes, cells can be left attached to the laminin-coatedplate for the following steps.

30. Add 550 μl fresh 18.5% formaldehyde or 625 μl of 16% formaldehyde to eachsample.

Formaldehyde is toxic, and the preparation process involving heating results in consider-able vaporization, which increases the hazard. Therefore, we suggest using commerciallyavailable 16% formaldehyde solution to reduce dangerous handling.

31. Incubate at room temperature with gentle shacking for 10 min.

32. Add 1 ml of 10× glycine to quench unreacted formaldehyde.

33. Swirl to mix and incubate at room temperature with gentle shaking for 5 min.

34. Place the samples on ice.

If working with adherent cardiomyocytes, scrape the cells and transfer into a 15-ml tube.

35. Centrifuge for 5 min at 800 × g, 4◦C, to pellet the cells.

36. Remove PBS/medium, being careful not to disturb the cells.

37. Add 10 ml cold 1× PBS (containing 1× protease inhibitor cocktail) to wash thecells.

38. Repeat steps 35 to 37.

39. Centrifuge for 5 min at 800 × g, 4◦C, to pellet the cells.

40. Remove PBS, being careful not to disturb the cells.

41. Proceed to the next step or snap-freeze with liquid nitrogen and store the cell pelletup to several months at −80◦C.

Lysis and sonication (1 hr)At this point more samples of the same origin can be combined (up to 3 × 106 cells).

42. Resuspend the cells in a 1.5-ml microcentrifuge tube (if frozen thaw cell pellet onice) in cell lysis buffer (100 to 500 μl, enough to cover the cell pellet and have aclear solution) containing protease inhibitor cocktail (PIC):

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100 μl cell lysis buffer + 0.5 μl PIC150 μl cell lysis buffer + 0.75 μl PIC200 μl cell lysis buffer + 1 μl PIC250 μl cell lysis buffer + 1.25 μl PIC500 μl cell lysis buffer + 2.5 μl PIC.

43. Incubate on ice for 15 min; vortex every 5 min.

44. Centrifuge for 5 min at 800 × g, 4◦C.

45. Remove the supernatant and resuspend the cells in a 1.5-ml microcentrifuge tube innuclei lysis buffer (100 to 500 μl, enough to cover the cell pellet and have a clearsolution) containing PIC. Incubate on ice 5 to 30 min.

100 μl nuclei lysis buffer + 0.5 μl PIC150 μl nuclei lysis buffer + 0.75 μl PIC200 μl nuclei lysis buffer + 1 μl PIC250 μl nuclei lysis buffer + 1.25 μl PIC500 μl nuclei lysis buffer + 2.5 μl PIC.

46. Sonicate the cell lysate on ice: 3 cycles of 7 min each, 30 sec on/30 sec off athigh power, using Bioruptor from Diagenode. If using a different device, sonicationmay require optimization. (Different samples of the same origin may be combinedtogether up to 500 μl total volume).

Keep the cell lysate cold. Sonication produces heat, which can denature the chromatin.

47. Centrifuge for 10 min at 14,000 × g, 4◦C.

48. Transfer the supernatant into fresh microcentrifuge tube. Make 50- to 200-μl aliquots(chromatin from 6 × 105 cells is sufficient for one assay). Proceed to the next stepor snap-freeze with liquid nitrogen and store the cells up to 2 months at −80◦C.

Perform agarose gel analysis of sonication (optional) (3 hr)See Critical Parameters and Troubleshooting for information on when to perform theseoptional steps.

49. Remove a 10- to 25-μl aliquot for agarose gel analysis.

50. Incubate for 10 min at 95◦C.

51. Add 1 μl RNase A (10 mg/ml) and incubate for 30 min at 37◦C.

52. Add 1 μl Proteinase K and incubate at 62◦C for 30 min to 2 hr with shaking.

53. Load 10 μl and 20 μl on a 1% agarose gel with a 100-bp DNA marker.

The majority of sheared chromatin should run between 200 and 1000 bp (Fig. 17.14.4).

Immunoprecipitate (30 min + overnight incubation)54. Remove PIC and thaw at room temperature. Prepare dilution buffer for the number

of samples and store on ice.

Remember to add a sample for negative control (Normal rabbit IgG); including a positivecontrol is also advisable.

55. Add dilution buffer to each sample as shown below:

If the volume of the sample is 50 μl: 450 μl dilution buffer and 2.25 μl PIC.If the volume of the sample is 100 μl: 900 μl dilution buffer and 4.5 μl PIC.If the volume of the sample is 200 μl: 800 μl dilution buffer and 4 μl PIC.

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1000 bp

500 bp

100 bp

Ladder Sample 1 Sample 2

Figure 17.14.4 Agarose gel analysis of sonicated DNA.

56. Add the dilution buffer into each tube.

57. Remove 5 to 10 μl (1%) of the supernatant from each sample as input and save at4◦C until step 63.

58. Add the proper antibody (5 μg in 500 μl total volume, 10 μg in 1000 μl; amountmay vary for different antibodies).

Polyclonal antibodies are preferable to monoclonal. Select ChIP-grade antibody whenavailable.

59. Incubate overnight at 4◦C with rotation.

Following day (3 to 4 hr):60. Add protein A magnetic beads (20 μl in 500 μl total volume, 30 μl in 1000 μl; make

sure that the beads are well mixed) to each sample and incubate for 2 to 3 hr at 4◦Cwith rotation.

Alternatively, protein G magnetic beads can be used: please consider the antibody classto select the protein with higher affinity.

61. Working at 4◦C, pellet magnetic beads with the magnetic separator and remove thesupernatant.

62. Working at 4◦C, wash the protein A-antibody-chromatin complex using 1 ml each ofthe cold buffers listed below. For each wash: add buffer, invert the tube to resuspendbeads, incubate 4 min on a rotating platform, centrifuge briefly, and place the tubein the magnetic rack. Wait 1 min and remove the supernatant.

a. Low salt wash buffer, two washes (can reduce to one wash if cell number is low)

b. High salt wash buffer, two washes (can reduce to one wash if cell number is low)

c. LiCl wash buffer, one wash

d. TE buffer, one wash.

Isolate the DNA (2 hr and 30 min)63. Set shaking incubator to 62◦C and heating block to 95◦C. Prepare elution buffer

for all sample tubes and input tubes: For each tube, 100 μl elution buffer + 1 μlProteinase K (300 μg/ml final).

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64. Add 100 μl elution buffer to each sample (include the input).

65. Incubate at 62◦C for 30 min to 2 hr with shaking.

66. Incubate for 10 min at 95◦C.

67. Pellet the magnetic beads with the magnetic rack and transfer the supernatant into afresh microcentrifuge tube.

Purify the DNA (30 min)68. Purify the DNA according to the DNA purification kit manufacturer’s guidelines

(e.g., Qiagen).

69. At the end, dilute the DNA with 30 μl prewarmed molecular grade water, centrifugeaccording to the manufacturer’s guidelines, and repeat (total volume = 58 μl).

Sample can be used immediately for analysis or can be frozen at −20◦C.

Perform real-time PCR (30 min preparation + 2 hr run)70. Design primers specific for analysis of the regions of interest.

Good primer design is a critical step: in general, primers should have a size of 20 to 24 bp,a Tm of 55◦ to 60◦C and a GC content of 50% to 60%. Amplification products should bebetween 150 to 250 bp. Primers should be tested on genomic DNA. Design of good primerscould be easily achieved using a free Web site like Primer3 (http://frodo.wi.mit.edu/).

71. Prepare the following mix for each sample (each sample should be run in triplicate):

2× Sybr green 7.5 μlPrimers (F+R) 10 μm 0.75 (500 nM final)DNA 3 to 4 μlWater up to 15 μl.

72. Start the following PCR protocol:

Initial cycle: 5 min 95◦C (initial denaturation)40 cycles: 15 sec 94◦C (denaturation)

30 sec 60◦C (annealing)30 sec 72◦C (extension).

Melting curve from 60◦C to 95◦C, read every 1◦C, hold 00:00:03.

Data analysisChIP-qPCR data need to be normalized for the different sources of variability. The twomost common methods are the percent input method (in which ChIP signals are dividedby input signal) and the fold enrichment methods (in which ChIP signal is presented asthe signal increase relative to background signal).

Percent input method method (Fig. 17.14.5A):

Calculate the percent input of each sample: IP sample = 2ˆ(Average Ct input−averageCt IP sample).

Fold Enrichment method (Fig. 17.14.5B):

Calculate the percent input of each sample: IP sample = 2ˆ(Average Ct IP sample–average Ct IgG).

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0

0.5

1

1.5

2

2.5A

0

1

2

3

44.5

B

% in

put

IgG IP sampleIgG IP sample

Fol

d en

richm

ent

3.5

2.5

1.5

0.5

Figure 17.14.5 Data analysis with (A) percent input method and (B) fold enrichment method.

REAGENTS AND SOLUTIONSUse deionized, distilled water in all recipes and protocol steps. For common stock solutions, seeAPPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.

Cardiomyocyte medium

For 500 ml of medium combine 500 ml DMEM or M199 with 500 mg bovineserum albumin (BSA), 500 mg 2,3 butanedione monoxime (BDM), 5 ml penicillin(10,000 U/ml)/streptomycin (10,000 μg/ml), and 5 ml insulin-transferrin-selenium-A. Filter-sterilize and store up to 3 months at 4◦C.

Cell lysis buffer

10 mM Tris·Cl, pH 8.010 mM EDTA0.5 mM EGTA0.25% Triton X-100

To prepare 200 ml cell lysis buffer, combine 2 ml of 1 M Tris·Cl, pH 8.0 (seerecipe), 4 ml of 0.5 M EDTA (see recipe), 200 μl of 0.5 M EGTA (see recipe), and5 ml of 10% of Triton X-100 in 190.8 ml H2O. Store up to 1 year at 4◦C.

Digestion buffer

Each heart will require 10 ml (prepare a separate 50-ml tube for each heart).Combine 10 ml perfusion buffer (see recipe), 40 mg collagenase D, 55.6 μl of 10×trypsin, and 1.25 μl of 100 mM CaCl2. Prepare fresh.

Dilution buffer

16.7 mM Tris·Cl, pH 8.01.2 mM EDTA167 mM NaCl0.01% SDS1.1% Triton X-100To prepare 200 ml dilution buffer, combine 4 ml of 1 M Tris·Cl, pH 8.0 (see recipe),

440 μl of 0.5 M EDTA (see recipe), 50 μl of 10% of SDS, 22 ml of 10% TritonX-100, and 8.36 ml of 4 M NaCl (see recipe) in 165 ml H2O. Store up to 1 yearat 4◦C.

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EDTA, 0.5 M

To prepare 0.5 M EDTA (pH 8.0), add 186.1 g disodium EDTA·2H2O to 800 mlH2O. Adjust the pH to 8.0 with NaOH (pellets). EDTA will not go into solutionuntil the pH of the solution is correct. Filter or autoclave to sterilize. Store up to1 year at 4◦C.

EGTA, 0.5 M

To prepare 10 ml solution, combine 1.9 g EGTA with 9 ml H2O. Adjust the pH to7.5/8.0 with NaOH (pellets) and adjust the volume to 10 ml with water. EGTA willnot go into solution without NaOH. Store up to 1 year at 4◦C.

Elution buffer

200 mM NaCl1% SDSTo prepare 50 ml elution buffer combine 5 ml of 10% SDS (see recipe) and 2.5 ml

of 4 M NaCl (see recipe) in 42.5 ml H2O. Store up to 1 year at 4◦C.

Formaldehyde, 18.5%

Combine 4.8 ml H2O with 0.925 g paraformaldehyde and 35 μl of 1 N KOH (seerecipe). Prepare fresh.

Glycine, 10×To prepare 200 ml glycine, dissolve 18.74 g glycine in 200 ml phosphate-bufferedsaline (PBS; Mediatech, cat. no. 21-030). Store up to 1 year at 4◦C.

High salt wash buffer

20 mM Tris·Cl, pH 8.02 mM EDTA500 mM NaCl0.1% SDS1% Triton X-100To prepare 200 ml high salt wash buffer, combine 4 ml of 1 M Tris·Cl, pH 8.0 (see

recipe), 800 μl of 0.5 M EDTA (see recipe), 2 ml of 10% SDS, 20 ml of 10%Triton X-100, and 25 ml of 4 M NaCl (see recipe) in 148.2 ml H2O. Store up to1 year at 4◦C.

KOH, 1 N

To make 10 ml, dissolve 0.56 g KOH in 10 ml H2O. Store up to 1 year at 4◦C.

LiCl, 1 M

Dissolve 4.24 g LiCl in a final volume of 100 ml H2O. Filter or autoclave to sterilize.Store the solution up to 1 year at 4◦C.

LiCl wash buffer

10 mM Tris·Cl, pH 8.01 mM EDTA0.25 M LiCl1% IGEPAL CA6301% deoxycholateTo prepare 200 ml LiCl wash buffer, combine 50 ml of 1 M LiCl (see recipe), 2 ml

of 10% IGEPAL CA630 (MP Biomedicals), 2 g of 10% of deoxycholate, 2 mlof 1 M Tris·Cl, pH 8.0 (see recipe), and 400 μl of 0.5 M EDTA (see recipe) in145.6 ml H2O. Store up to 1 year at 4◦C.

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Low salt wash buffer

20 mM Tris·Cl, pH 8.02 mM EDTA150 mM NaCl0.1% SDS1% Triton X-100To prepare 200 ml low salt wash buffer, combine 4 ml of 1 M Tris·Cl, pH 8.0 (see

recipe), 800 μl of 0.5 M EDTA (see recipe), 2 ml of 10% SDS, 20 ml of 10%Triton X-100, and 7.5 ml of 4 M NaCl (see recipe) in 165.7 ml H2O. Store up to1 year at 4◦C.

NaCl, 4 M

Dissolve 23.37 g NaCl in 10 ml H2O. Filter or autoclave to sterilize. Store up to1 year at 4◦C.

Nuclei lysis buffer

50 mM Tris·Cl, pH 8.010 mM EDTA1% SDSTo prepare 200 ml nuclei lysis buffer, combine 10 ml of 1 M Tris·Cl, pH 8.0 (see

recipe), 4 ml of 0.5 M EDTA (see recipe), and 20 ml of 10% SDS (see recipe)in 166 ml H2O. Store up to 1 year at 4◦C.

Perfusion buffer, 1×To prepare 500 ml of buffer (enough for five to seven isolations), combine 3.515 gNaCl, 0.55 g KCl, 0.041 g KH2PO4, 0.0425 g Na2HPO4, 0.15 g MgSO4·7H2O, 1.3g Na-HEPES, 0.195 g NaHCO3, 1.875 g taurine, 0.5 g BDM, and 0.5 g D-glucose.Adjust the pH to 7.4 and then filter the solution. Prepare fresh. If multiple isolationswill be performed in a week, a 10× perfusion buffer can be prepared and stored upto 1 week at 4◦C. In this case, prepare the 10× buffer without taurine, BDM, andD-glucose. These will be added fresh to the 1× buffer on the day of the isolation.

SDS, 10%

Dissolve 10 g SDS in 100 ml H2O. Store up to 1 year at 4◦C.

Stop buffer A

Each heart will require 10 ml. Combine 9 ml perfusion buffer (see recipe), 1 mlfetal bovine serum (FBS), and 1.25 μl of 100 mM CaCl2. Prepare fresh.

Stop buffer B

Each heart will require 10 ml. Combine 9.5 ml perfusion buffer (see recipe), 0.5 mlfetal bovine serum (FBS), and 1.25 μl of 100 mM CaCl2. Prepare fresh.

Tris·Cl, pH 8.0, 1 M

Dissolve 121.1 g Tris base in 800 ml H2O. Adjust the pH using HCl, and then adjustthe volume of the solution to 1 liter with H2O. Filter or autoclave to sterilize. Storeup to 1 year at 4◦C.

COMMENTARY

Background informationChIP is a type of immunoprecipitation used

to investigate the interaction between proteinsand DNA in vivo. There are two main types

of ChIP: the cross-linked ChIP, which re-versibly cross-links (with formaldehyde or UVlight) the sheared chromatin (usually by soni-cation), and the native ChIP, which uses native

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chromatin sheared by micrococcal nucleasedigestion. The cross-linked ChIP is the mostcommon method for the analysis of DNA tar-get of transcription factors or other chromatin-associated proteins.

The first reports of cross-linking ChIPfor chromatin in vivo studies date back tothe 1980s when Gilmour and Lis (Gilmourand Lis, 1984), and Varshavsky and Solomon(Solomon and Varshavsky, 1985), publishedtheir pioneering work in the field of chromatinimmunoprecipitation. The two studies differedin that the cross-linking method of Gilmourand Lis utilized UV irradiation to cross-link proteins with neighboring DNA whereasVarshavsky and Solomon used formaldehydecross-linking.

It is worth mentioning that since its dis-covery ChIP has constantly evolved and many“flavors” of ChIP have been elaborated (Col-las, 2009). For instance, ChIP–reChIP takesadvantage of two sequential immunoprecipi-tations to determine whether two or more pro-teins are close enough in a chromatin contextor whether they are interacting, probably form-ing a complex, in a localized genomic region(Truax and Greer, 2012). More importantly,chromatin immunoprecipitation, followed bymicroarray hybridization (ChIP-chip) or bynext-generation sequencing analysis (ChIP-Seq), is currently an invaluable method withwhich to investigate the genome-wide distribu-tion of chromatin-binding proteins and histonemodifications in any genome with a known se-quence. A detailed description of these tech-niques is beyond the scope of this unit, andwe refer the reader to exhaustive and updatedreviews (Park, 2009; Kidder et al., 2011).

Epigenetics may be defined as the studyof any potentially stable, and ideally heritablechange in gene expression or cellular pheno-type that occurs without changes in Watson-Crick base pairing of DNA (Goldberg et al.,2007). Much of today’s epigenetic research isconverging on the study of covalent and nonco-valent modifications of DNA and histone pro-teins, and the mechanisms by which such mod-ifications influence overall chromatin structure(Bernstein and Hake, 2006; Goldberg et al.,2007). Since histones and histone PTMs arecurrently taking the center stage of epigeneticcontrol, ChIP has become the election tech-nique in the epigenetics world.

A number of recent studies highlight therole of epigenetic regulation as a key pro-cess in mechanisms of cardiovascular devel-opment and disease. The importance of theepigenetic regulatory mechanisms for tran-

scriptional control in the cardiovascular sys-tem is just beginning to be appreciated, andgrowing evidence demonstrates that abnormalepigenetic regulation contributes to heart dis-ease (Wamstad et al., 2012). Therefore, inthe last year we have noted increasing inter-est amongst cardiovascular scientists in epige-netic studies along with their associated com-plex methodological challenges. For example,it was recently reported that the Polycomb Re-pressive Complex 2 (PRC2) regulates normalmouse heart formation through its H3K27-methyltrasferase component Ezh2 (He et al.,2011; Delgado-Olguin et al., 2012). Jarid2,another component of PRC2, is also knownto be fundamental during heart development(Lee et al., 2000). Brg1/Brm-associated factor(BAF) complexes also have important rolesin regulating gene expression during heart de-velopment (Takeuchi et al., 2011). Moreover,Wamstad et al. found that, during cardiac dif-ferentiation, distinct chromatin patterns arecoordinated with stage-specific expression offunctionally related genes. They also identifystage-specific enhancers and DNA-bindingmotifs within these regions that predict setsof transcription factors that orchestrate cardiacdifferentiation (Wamstad et al., 2012).

Adult cardiomyocytes are primary cells,which are fundamental for many studies. Theirimportance is further underscored by the factthat no cell lines are available with the sameproperties. Cell lines divide in culture whereasa defining characteristic of cardiomyocytes isthat they proliferate throughout fetal develop-ment and into the early neonatal period whendivision ceases and DNA replication declinesquickly (Parker and Schneider, 1991; Brookset al., 1998). Isolation and culturing of car-diomyocytes is a delicate process and the pro-tocol presented here is a modification of a pro-cedure presented by O’Connell et al. (2007),optimizing the combination of cardiomyocyteisolation with ChIP. The enzymes utilized forthe isolation of cardiomyocytes (collagenasein this protocol) are likely not altering the hi-stone proteins within the nuclei if the cell iso-lation is yielding intact cells. One cannot beabsolutely certain without analyzing histonemodifications in isolated cardiomyocytes andcomparing them to histone modifications inintact cardiac tissue. This is difficult to do, ascardiac tissue contains many cell types in ad-dition to cardiomyocytes, and this analysis hasnot been reported to our knowledge.

This unit describes the isolation and theChIP protocol for adult cardiac myocytes.However, this method could be applied to

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other tissue-derived cells. In fact, this methodprovides modifications that are applicable toworking with primary cells, in which cell num-ber is a very significant limitation. Some cell-and tissue-specific modifications may be re-quired, especially for the isolation procedureand in order to optimize the conditions forshearing cross-linked DNA.

Critical Parameters andTroubleshooting

Cardiomyocyte isolation: To obtain largenumbers of healthy cardiomyocytes, all of thesteps described above are critical and it is usu-ally necessary to perform several experimentsfor practice. In general, when working withheart tissue and cardiomyocytes, always workfast but very gently and avoid vigorous agita-tion. In particular, cannulation should be doneas quickly as possible (1 minute is desirable)(O’Connell et al., 2007); collagenase shouldbe tested frequently to assess lot-to-lot consis-tency; and cardiomyocytes should attach to thelaminin-coated plate very quickly (less than 30minutes). If this does not happen, prepare newlaminin-coated plates and transfer the cells.

Cardiomyocyte cross-linking: In this steptwo factors are critical: formaldehyde has tobe high quality and freshly prepared and ex-tent of the cross-linking has to be accurate.Histones are tightly associated with DNA andexcessive cross-linking can modify the ly-sine residues in their tails. Moreover, cellsthat are over-cross-linked will not producesmall chromatin fragments, even by prolongedsonication, therefore resulting in lower yield(Orlando et al., 1997).

Lysis and sonication: It is very important tohave efficient lysis. For this, make sure thereis enough lysis buffer. Flash-freezing the cellsafter the incubation in the nuclei lysis buffercan help open the nuclei more efficiently. Son-ication is also a critical step: perform it in acold room and refill the water bath with iceevery 5 to 7 min (or every time that the waterbecomes warm). Shearing the DNA to a smallsize (between 100 to 1000 bp) is an impera-tive step to achieve good resolution. Becauseof the importance of this step, analysis of thesheared chromatin by gel electrophoresis isstrongly recommended (steps 49 to 53), es-pecially when setting up the protocol and foreach different cell type.

Immunoprecipitation: The quality of anti-bodies used for ChIP is one of the most im-portant factors that contribute to the qualityof the data generated by these studies. High-sensitivity and specificity antibodies are re-

quired because they allow the detection ofenrichment without substantial backgroundnoise. Many commercial antibodies that havebeen tested for their use in ChIP studiesare available. However, results from variousgroups have shown that not all commercialantibodies that are designated as “ChIP grade”or “ChIP qualified” can be successfully used.Furthermore, commercially available ChIP-grade antibodies suffer from lot-to-lot varia-tion, leading to nonspecific binding and ques-tionable datasets (Egelhofer et al., 2011; Kid-der et al., 2011; Peach et al., 2012). Whenpossible, use polyclonal and ChIP-grade anti-bodies. When choosing antibodies, check theliterature to select antibodies that have beensuccessfully used for ChIP experiments. In ad-dition, make sure that the subclass and the iso-type of the antibody are correct for the protein(A/G) magnetic bead used (in general, rab-bit antibodies work for both A and G pro-teins while certain classes of mouse and ratimmunoglobulins do not bind with protein A).

Real-time PCR: For successful PCR, thechoice of primers is essential (see step 59). Insome cases, it may be necessary to increasethe amount of DNA added to the reaction. Ingeneral, standard PCR parameters and trou-bleshooting should be followed.

Anticipated ResultsChIP experiments usually require at least

106 cells for each immunoprecipitation. Thisprotocol aims to increase the yield of the car-diomyocyte isolation while reducing the num-ber of cells required to perform the ChIP exper-iments. Therefore, we anticipate this methodwill optimize buffer, decrease number of stepsand timing, keep the procedure simple andstraightforward, and enable immunoprecipita-tion with a smaller number of cells than usuallyrequired for standard ChIP protocols.

Time ConsiderationsThe entire protocol can be completed in

2 days. On the first day, cardiomyocytes canbe isolated (approximately 2 to 3 hr for onemouse) and then chromatin can be cross-linked(less than 2 hr), sonicated (1 hr + 3 hr forgel analysis), and incubated overnight with theantibody of interest. The following day DNAcan be washed, isolated, and purified (roughly6 hr). Following this, quantitative PCR can beperformed (3 hr).

For convenience, one whole day can be ded-icated to cardiomyoyte isolation (five mice ormore), and the cells can be snap frozen and

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stored at −80◦C. ChIP protocol (2 days) canthen performed at a convenient time.

At several points during this procedure (in-dicated in the above protocol) one may chooseto store the samples and stop the experiment.

AcknowledgementsThis work was supported by NIH grant

(R01-HL 088255).

Literature CitedBernstein, E. and Hake, S.B. 2006. The nucle-

osome: A little variation goes a long way.Biochem. Cell Biol. 84:505-517.

Brooks, G., Poolman, R.A., and Li, J.M. 1998.Arresting developments in the cardiac myocytecell cycle: Role of cyclin-dependent kinase in-hibitors. Cardiovasc. Res. 39:301-311.

Collas, P. 2009. The state-of-the-art of chro-matin immunoprecipitation. Methods Mol. Biol.567:1-25.

Delgado-Olguin, P., Huang, Y., Li, X.,Christodoulou, D., Seidman, C.E., Seid-man, J.G., Tarakhovsky, A., and Bruneau,B.G. 2012. Epigenetic repression of cardiacprogenitor gene expression by Ezh2 is requiredfor postnatal cardiac homeostasis. Nat. Genet.44:343-347.

Donovan, J. and Brown, P. 2006. Euthanasia. Curr.Protoc. Immunol. 73:1.8.1-1.8.4.

Egelhofer, T.A., Minoda, A., Klugman, S.,Lee, K., Kolasinska-Zwierz, P., Alekseyenko,A.A., Cheung, M.S., Day, D.S., Gadel, S.,Gorchakov, A.A., Gu, T., Kharchenko, P.V.,Kuan, S., Latorre, I., Linder-Basso, D., Luu, Y.,Ngo, Q., Perry, M., Rechtsteiner, A., Riddle,N.C., Schwartz, Y.B., Shanower, G.A., Vielle,A., Ahringer, J., Elgin, S.C., Kuroda, M.I., Pir-rotta, V., Ren, B., Strome, S., Park, P.J., Karpen,G.H., Hawkins, R.D., and Lieb, J.D. 2011.An assessment of histone-modification antibodyquality. Nat. Struct. Mol. Biol. 18:91-93.

Gilmour, D.S. and Lis, J.T. 1984. Detectingprotein-DNA interactions in vivo: Distribu-tion of RNA polymerase on specific bacterialgenes. Proc. Natl. Acad. Sci. U.S.A. 81:4275-4279.

Goldberg, A.D., Allis, C.D., and Bernstein, E.2007. Epigenetics: A landscape takes shape. Cell128:635-638.

He, A., Ma, Q., Cao, J., von Gise, A., Zhou, P.,Xie, H., Zhang, B., Hsing, M., Christodoulou,D., Cahan, P., Daley, G.Q., Kong, S.W., Orkin,S.H., Seidman, C.E., Seidman, J.G., and Pu,W.T. 2011. Polycomb repressive complex 2 reg-ulates normal development of the mouse heart.Circ Res. 110:406-415.

He, A.B., Shen, X.H., Ma, Q., Cao, J.J., von Gise,A., Zhou, P.Z., Wang, G., Marquez, V.E., Orkin,S.H., and Pu, W.T. 2012. PRC2 directly methy-lates GATA4 and represses its transcriptional ac-tivity. Gene Dev. 26:37-42.

Kidder, B.L., Hu, G., and Zhao, K. 2011. ChIP-Seq:Technical considerations for obtaining high-quality data. Nat. Immunol. 12:918-922.

Lee, S., Lee, J.W., and Lee, S.K. 2012. UTX, ahistone H3-lysine 27 demethylase, acts as a crit-ical switch to activate the cardiac developmentalprogram. Dev. Cell 22:25-37.

Lee, Y., Song, A.J., Baker, R., Micales, B., Conway,S.J., and Lyons, G.E. 2000. Jumonji, a nuclearprotein that is necessary for normal heart devel-opment. Circ. Res. 86:932-938.

O’Connell, T.D., Rodrigo, M.C., and Simpson, P.C.2007. Isolation and culture of adult mouse car-diac myocytes. Methods Mol. Biol. 357:271-296.

Orlando, V., Strutt, H., and Paro, R. 1997. Analysisof chromatin structure by in vivo formaldehydecross-linking. Methods 11:205-214.

Park, P.J. 2009. ChIP-seq: Advantages and chal-lenges of a maturing technology. Nat. Rev.Genet. 10:669-680.

Parker, T.G. and Schneider, M.D. 1991. Growth-factors, protooncogenes, and plasticity of thecardiac phenotype. Annu. Rev. Physiol. 53:179-200.

Peach, S.E., Rudomin, E.L., Udeshi, N.D., Carr,S.A., and Jaffe, J.D. 2012. Quantitative assess-ment of ChIP-grade antibodies directed againsthistone modifications reveals patterns of co-occurring marks on histone protein molecules.Mol. Cell Proteomics 11:128-137.

Sdek, P., Zhao, P., Wang, Y.P., Huang, C.J., Ko,C.Y., Butler, P.C., Weiss, J.N., and MacLellan,W.R. 2011. Rb and p130 control cell cycle genesilencing to maintain the postmitotic phenotypein cardiac myocytes. J. Cell Biol. 194:407-423.

Solomon, M.J. and Varshavsky, A. 1985.Formaldehyde-mediated DNA proteincrosslinking—a probe for in vivo chro-matin structures. Proc. Natl. Acad. Sci. U.S.A.82:6470-6474.

Takeuchi, J.K., Lou, X., Alexander, J.M., Sugizaki,H., Delgado-Olguin, P., Holloway, A.K., Mori,A.D., Wylie, J.N., Munson, C., Zhu, Y., Zhou,Y.Q., Yeh, R.F., Henkelman, R.M., Harvey, R.P.,Metzger, D., Chambon, P., Stainier, D.Y., Pol-lard, K.S., Scott, I.C., and Bruneau, B.G. 2011.Chromatin remodelling complex dosage modu-lates transcription factor function in heart devel-opment. Nat. Commun. 2:187.

Truax, A.D. and Greer, S.F. 2012. ChIP and Re-ChIP assays: investigating interactions betweenregulatory proteins, histone modifications, andthe DNA sequences to which they bind. MethodsMol. Biol. 809:175-188.

Wamstad, J.A., Alexander, J.M., Truty, R.M.,Shrikumar, A., Li, F., Eilertson, K.E., Ding,H., Wylie, J.N., Pico, A.R., Capra, J.A., Er-win, G., Kattman, S.J., Keller, G.M., Srivastava,D., Levine, S.S., Pollard, K.S., Holloway, A.K.,Boyer, L.A., and Bruneau, B.G. 2012. Dynamicand coordinated epigenetic regulation of devel-opmental transitions in the cardiac lineage. Cell151:206-220.