Chip Protocol modified from Miles Browns lab(with tips from Direnzo and Shang)
This initial copy is very detailed and at the end is a working copy that you can take into
the lab with you
Preparation of pipettes and solutions
ChIP uses diagnostic PCR, and as with all diagnostic PCR protocols you have to be
aware of the possibility of contamination by pieces of DNA that will be amplified by
your primers. This could be a plasmid containing the fragment, or the PCR fragment
itself (from a previous experiment). This contamination comes from the inside of
Gilson pipettes - tiny amounts of DNA are sucked up while you are pipetting (any
movement of liquid creates aerosols). All of your reagents (including the stock solutions
you use to make them, e.g. water, Tris, EDTA etc) should be made up using stocks that
have never seen Gilson pipettes that may be dirty.
One of the defenses you have are plugged tips. But remember, these are irrelevant if the
solution you’re transferring already has some contamination in it.
The best thing is to make up or obtain completely fresh and separate reagents for
ChIP.
Note – this includes things you might not initially think of, like the phenol, the bead
slurries you use in the IP, the proteinase K etc. Make extra boxes and keep these
solutions/reagents hidden from everyone else eg in a drawer, or in the fridge/freezer in a
clearly marked box. These reagents should only be transferred using a clean Gilson
pipette or, for larger volumes, a disposable pipette (eg plastic 5 ml).
Gilson pipettes should be thoroughly cleaned before you make up solutions, and before
you do the ChIP assay itself.
Cleaning Gilson pipettes:
Disassemble the pipettor. Scrub the metal and rubber parts with detergent, rinse
thoroughly. The plastic barrel can be soaked in 0.25M HCl for ~20-30 minutes to
depurinate DNA. Wash thoroughly in soap and water, then rinse. You can do a final
rinse with EtOH to speed up the drying process (don’t use EtOH that’s seen a Gilson
pipette though!). NOTE – some idiots think that DNA goes away if you wash with
EtOH – it does not – in fact EtOH precipitates DNA, so if there is any on the barrel,
EtOH will do nothing to get rid of it.
Once you’ve cleaned your pipettes make sure no-one else uses them (eg put a notice on
your bench that your doing a ChIP assay) because their solutions may be contaminated
with DNA fragments that will screw up your ChIP assay.
(they use MCF-7 cells and then treat with -estadiol)
I have used several cell types now and the cross-link time seems to be the same. Going
by what others groups say specific transcription factors may require up to 30 min to
efficiently cross-link. The number of input cells seems to make a difference. I was able
to chip efficiently with 3 X 106 cells per I.P. but Peggy Farnham’s groups claims that
they need 1 X 107 –2 X 107 cells per I.P. to get a significant specific over background
signal.
1. Cross-link cells 10 min at room temp in media with 1% formaldehyde
(ie for 9 mL media use 243 uL of 37% formaldehyde).
2. Rinse cells twice with ice cold PBS and collect in 1 mL ice cold PBS
3. Spin down and wash once with ice cold PBS
4. Cells were then resuspended in 900uL of lysis buffer. Add Protease inhibitors
(PMSF, leupeptin, pepstatin A Stock made to 1000X and uL used).
Note – add the protease inhibitors just before you need the buffer. These are important –
eg histone H3 N-terminus is very protease sensitive – despite cross-linking, wise to add
these.
Note – PMSF stocks should be re-made monthly. Add shortly before use to lysis buffer –
PMSF half life is 30min in aqueous solution
Incubated 10 min on ice followed by sonication 3X for 15 s each at maximum setting.
Set aside 40 uL of this chromatin in a separate tube and freeze at –80 C, this will be used
later as the input sample for the PCR. Take a 10 uL(or 30uL) chromatin sample and add
2µl( or 6uL) of 5 M NaCl and reverse the crosslinks overnight at 65 C. Phenol
chloroform extract, precipitate, dry and resuspend in water and run about a quarter of the
sample on a 2-3% agarose gel to ascertain the degree of sonication
Phenol chloroform extraction after reverse crosslinking:
-add 100 ul of TE
-extract with 2X Phenol/Chloroform/Isoamyl alcohol and vortex well
-spin down 5 min r/t at max speed
-take upper aqueos phase and add to 2X 100% EtOH
-incubate 15 min on ice
-spin 20min at 4 C
-wash with 500 ul of 70% EtOH spin 5 min at 4 C
-speed vac 10 min
-resuspend in 20 ul of TrisHCl pH
- run out 15 ul and 5 ul on a 1.5- 2.0% agaose gel and visualized
sonication
This sonication step needs to be determined for each cell line you use. I generally make a
stock chromatin of 3 X 107 cells in 1mL of lysis buffer(this is enough cells for 10 IP’s).
This way I always do my sonications in the same volume. This is necessary as the
volume of the sonication and the number of cells in the soication will both affect how
much sonication is required for your specific cell line. Often the literature says that
chromatin was sonicated to an average size of 500 bp. Talking to the ChiP people we
discovered that often larger fragments ie 1000-1500 give a better chip result.
5. Centrifuge at max speed for 10 min at 4 C(this gets rid of any of the insoluble cellular
crap).
6. Collect the supernatant in a new tube and dilute in IP dilution buffer 1:10. The
chromatin suspension must be diluted at least 1:10 to reduce to overall SDS concentration
to less than 0.1%.
7. Pre-clear with 2ug of salmon sperm DNA and protein A-Sepharose(45 uL of 50%
slurry in 10 mM Tris-HCl(pH 8.1) 1mM EDTA) for 2hr at 4ºC. Do a quick spin and
collect the supernatant.
8. Immunoprecipitate 6hr or overnight at 4ºC rocking. The amount of antibody required
needs to be determined usually by standard IP optimization test.
9. After immunoprecipitation spin at 4ºC for 10 minutes to get rid of insoluable junk,
and transfer supernatant to a new tube. This is important as in subsequent steps this junk
will pellet with your bead complex. Now add 45 uL of protein A-Sepharose and 2 ug of
salmon sperm DNA were added and the incubation continued for 1 hr
Preparing the Beads
Make sure bead stock is mixed well (it’s a suspension, not a solution), then
take as much as you need (20ul per IP) plus one half extra (eg if you have 3
samples take 3.5 x 20ul = 70ul). Mark the tube at the level of the meniscus.
Wash 3x with IP dilution buffer. After the last wash, remove buffer to the
level of the mark, then add an equal volume (e.g. 70ul) of 10mg/ml tRNA
10. Precipitates were washed sequentially with 1 mL each of the following for ten
minutes:
(a) 1X TSEI
(b) 4X TSEII (this wash is important to get rid of background/ non specific DNA)
(c) 1X Buffer III
11. The precipitates were then washed 3X with 1 mL of TE( this wash important to
reduce background)
12. Then samples are extracted two times with 250 uL extraction solution. Extraction is
done by vortexing samples 30s or longer and then rotating for 5 min. This is done twice
collecting in a new tube each time.
13. Eluates were combined and heated at 65ºC for at least 6 hr to reverse cross links.
Take the 40 uL of the input chromatin you saved at the lysis step and add 8 ul of 5M
NaCl and incubate at least 6 hr with at the same time as the IP samples.
14. Samples were purified using the Qiaex II gel extraction/concentration kit. This step
needs to be done in 2 mL microcentrifuge tubes and generally I use 20-30uL of beads per
IP sample. For the input samples (ie only 40uL) add 400 uL of 10mM Tris/HCl and
extract as with the other samples. The kit is much faster and if supposed to give a higher
recovery than phenol/chloroform extraction.
15. 1-5 ul of 35 ul was used to PCR amplify. I have generally been using 3 uL in a 50 uL
reaction. For the input DNA I dilute it 1:10 in Tris/HCl and then use 3 uL of this in a 50
uL PCR reaction. I then run out 40 uL of the IP PCR reactions and the input PCR
reaction. As a positive control for the PCR reaction I use 100 ng of genomic DNA in a
50 uL reaction and only run out 20 uL. Of course you want to have a negative control for
PCR where no DNA is added.
As far as experimental design goes reactions will include your target promoter(s) and at
least one control region of DNA that does not bind the txn factor you’re studying; or, if
its histone acetylation changes your studying, you need a control where acetylation
doesn’t change (e.g a constitutively active promoter region). Good acetlylation controls
are the mouse histone H4 promoter(used in Peter Cheungs Mol Cell pater) and the human
Gamma Actin promoter.
Another important aspect of the ChiP assay is having the diagnostic PCR product you
generate lie in the linear range of amplification. If the number of cycles you use to PCR is
to high you will reach a point where linear amplification will cease occur( See picture
below). This is a problem if you are trying to compare the same PCR reaction done on
two different IP samples because if you are out of the linear range you may miss
quantitative difference in the amount of DNa IP’d. If you are trying to compare
diagnostic PCR reactions across a whole region you obviously will be comparing
different PCR reactions and therefore different kinetics of amplification. Here the
analysis required for amplification is a little more complex but can be done. Read below
to see how this is done.
IP Antibody
No AB Brg-1 Gal4
o
Ab
26
cycles
No AB Brg-1 Gal4 No AB Brg-1 Gal4
28
30
6000
No AB Brg-1 Gal4 No AB Brg-1 Gal4
32
34
cycles
cycles
5000
IP Antibody
Volume
4000
none
3000
brg-1
gal4
2000
1000
0
30
32
34
Number of Cycles
Steps to take when trying to quatify ChIP:
The first step to doing a good ChIP is to have the diagnostic PCR your are using as highly
optimized as possible. Do a range of annealing temperatures and salt conditions. I
usually try 3 annealing temperatures(over about 5-10 degeree range), and three different
salts(1mM, 2mM, and 3mM). Once you have a good PCR reaction you can begin to
analyse ChIP’d DNA
Method A: If you are only looking at one loci
This is simple because you only need to account for the efficiency of one PCR and
therefore only determine the linear range for your IP’d sample.
1. Set up your initial PCR with 34 cycles under you optimized PCR conditions.
-this will most likely be out of the linear range but it will ensure that you won’t
miss any potential signal.
2. Run out you PCR product and determine if the IP sample contains an IP’d band.
3. To ensure that you have a product that is in the linear range set up a titration of the
PCR varying the number of cycles..
-at the initial higher cycle number you may get a lower signal to noise ratio as
your diagnostic PCR has ceased to amplify linearly but the background signal is
still amplifying in a linear fashion
-decreasing the number of cycles you should be able to observe a cycle range
where the volume of the PCR product doubles with every cycle
-on our machine you have to manually remove the samples at your desired cycle
-this is done by setting up the normal cycle you would use for you amplification.
-you can see how many cycles are completed on your block by selecting the
active block and pushing the arrow button down. When you reach the point
where the 27th cycle has completed you need to watch the cycle. First let the
denature step complete, followed by the annealing cycle. Then transfer the tubes
to a second block holding at 72 C and allow the final extension to go for ten
minutes. You have now collected a sample that has completed 28 cycles. This
same method is then used to collect samples over your desired range of cycles.
3. Run out your samples on an agarose gel and use the volume tool to determine the
amount of each product you have generated. You should be able to plot a titration similar
to the one I showed above.
4. Once you determine the number of cycles that amplifies your IP’d product in the
linear range include a dilution of your input DNA that also demonstrated linear
amplification in a range of similar intensity to your IP’d band.
-this kind of assay essentially is just to give you a “yes” or “no” answer as to
whether your given factor binds the diagnostic region.
-being in the linear range assures you that you have a significant signal to noise
ratio.
Method B: If you are trying to observe quantitative differences between several loci that
are closely associated(ie acetylation across a several kb region)
**Papers you must read before attempting this**
Science 2001 Aug 10;293(5532):1150-5
Transitions in distinct histone h3 methylation patterns at the heterochromatin domain boundaries.
Noma Ki, Allis CD, Grewal SI.
EMBO J 2001 May 1;20(9):2224-35
Transitions in histone acetylation reveal boundaries of three separately regulated neighboring loci.
Litt MD, Simpson M, Recillas-Targa F, Prioleau MN, Felsenfeld G.
Proc Natl Acad Sci U S A 2000 Aug 29;97(18):10014-10019
Histone deacetylase inhibitor selectively induces p21WAF1 expression and gene-associated histone
acetylation.
Richon VM, Sandhoff TW, Rifkind RA, Marks PA.
For this type of analysis some groups use and internal control PCR (Grewal, Allis) and
other groups don’t(Felsenfeld, Marks). The internal control is to account for differences
in pipetting DNA and loading and PCR reactions. If you choose to use an internal control
it should be something that will not vary quantitatively in your IP’d samples. For
example in mouse an internal control for acetylation is histone H4. For this method you
need to optimize a multiplex PCR reaction. This will give you the ability to normalize
any diagnostic PCR product against the internal control to account for pipetting DNA and
loading sample.
1. Once you have optimized you PCR conditions for each set of primers you need to
roughly normalize for differences in efficiency between your PCR reactions.
-This can be tested on input DNA that was used in your IP. Your need to first
determine with a set amount of DNA(ie something similar to what you expect in
the IP sample determined empirically) what the linear range of your PCR is by
doing varying numbers of PCR cycles.
- Once you get your linear range determined you will use this as a reference point
to set up input titration’s that will be used to ensure the PCR of your IP’d reaction
is within the linear range of amplification.
2. Now that you are in the correct range do a small test with your IP samples varying the
number of cycles to determine on the IP sample that you generated will fall in the linear
range.
3. Once you are confident that you can amplify (A) the PCR in a linear number of cycles
and (B) that the amount of IP DNA will amplify within this range, for each desired
diagnostic PCR you can then PCR the whole range of samples in a quantitative fashion.
5. The analysis then is where theoretical considerations come in. Despite that fact that
you have generated PCR diagnostics that are in the linear range (and you may have an
internal control to ensure similar loading etc), you have not really accounted for the
differences in PCR efficiency between primer pairs. If you don’t account for this you
will fool yourself into believing quantitative difference across a locus occur when really
it is just a reaction efficiency difference. This normalization is done as Grewals group
did in their yeast histone methylation paper. Using the input(or as they call it the Whole
Cell Extract(WCE)) and do the same multipex PCR under the same reaction condtions.
Then by comparing the ((IP band)/(IP internal control band))/((Input band)/Input internal
control band)) you get a value which is the relative enrichment at a given locus. This
analysis is probably tougher in mammalian cells than in yeast but it is necessary to get
useful data. If you don’t have an internal pipetting DNA/ loading control you are
making the assumption that you added the same amounts of DNA and loaded equal
amounts of PCR product. This shouldn’t be a real issue as you repeat your experiment
you will gain numerical data and standard deviation values that will tell you if a given
experiment is flawed and represents an outlier. This analysis is much simpler you can
just look at your given loci dividing the amount of amplified IP signal to that of the input
sample. So for our experiments you would have ((induced IP)/(induced
input))/((uninduced IP)/(uninduced input)). This would give you a fold enrichment at a
given loci.
Solutions:
Lysis Buffer
500 mL
1% SDS
10mM EDTA
50mM Tris-HCl(pH to 8.1)
add protease inhibitors(fresh)
50mL of 10% SDS
5 mL of 1 M EDTA
25 mL of Tris-HCl (pH 8.1)
IP dilution buffer
500 mL
1% tritonX-100
2mM EDTA
150mM NaCl
20 mM Tris-HCl(pH to 8.1)
5mL of 100% Triton
1mL of 1 M EDTA
15 mL of 5 M NaCl
10 mL of 1M Tris-HCl(pH to 8.1)
TSE I(very similar to IP dilution buffer in old protocol)
500 mL
0.1 % SDS
5 mL of 10% SDS
1% TritonX-100
5mL of 100% Triton
2mM EDTA
1mL of 1 M EDTA
20mM Tris-HCl(pH 8.1)
10 mL of 1M Tris-HCl(pH to 8.1)
150mM NaCl
15 mL of 5 M NaCl
TSE II( same as tse 500 in old protocol)
0.1 % SDS
1% TritonX-100
2mM EDTA
20mM Tris-HCl(pH 8.1)
500mM NaCl
Buffer III(Similar to detergent wash buffer in old protocol)
500 mL
0.25 M LiCl
125 mL of 1 M LiCl
1% NP-40
50 mL of 10% NP40
1% deoxycholate
50 mL of 1 % deoxycholate
1mM EDTA
500 uL of 1M EDTA
10mM Tris-HCl(pH 8.1)
5 mL of 1M Tris-HCl(pH to 8.1)
Extraction Solution(same as old protocol)
1% SDS
0.1M NaHCO3
Protease Inhibitors
100 mM PMSF in ethanol, use at 1:100
10 mg per ml aprotinin in 0.01 M HEPES pH 8.0, use at 1:1,000
10 mg per ml leupeptin in water, use at 1:1,000
Fig * Factors that affect the efficiency of detection in the ChIP protocol
In designing the ChIP experiments, we considered the variables that affect efficiency of detection, and derived
a simple formula that estimates the fraction of sonicated fragments containing a binding site that will be
detected by ChIP for any set of sonication and PCR criteria.
Definition of symbols:
F = Fraction of fragments bound to factor X that can be detected by ChIP
S = Average Size of sonicated fragments.
P = size of diagnostic PCR fragment
E = size of Element that binds factor X.
I = Interval between end of PCR fragment and binding site
T = Total size of interval P + E + I (T = P if binding site and PCR fragment overlap).
If S = 1500bp, the number of sonicated fragments that contain at least one bp of a 1500bp region is 1500
(assuming that sonication occurs equally at every base*). The number of these fragments that include the
10bp factor X binding site is S-E (1490).
Of the same 1500 fragments, the number that include the diagnostic PCR fragment and the binding site for X
is S-T.
So, the fraction of all immunoprecipitated fragments that will contain the diagnostic PCR region is obtained
by:
F=
S-T
S-E
Consider the example:
Size
(bp)
X is present on
1490 1500bp fragments
300
400
1
0
Factor
X
PCR
primers
1500 bp sonicated
fragments that bind X
Thus
S = 1500
P=300, E = 10, I=400
..so T=710
and F = 0.53
In other words, 53% of all the fragments that bind X can beamplified. The PCR diagnostic would detect binding of
X at this frequency 400bp 5’ or 3’ of the end of the PCR fragment. Thus, under these conditions, a total region of
1420bp (2 x T) is covered at 53% efficiency. When the binding site is within the PCR fragment T = P so F =0.8.
So at best, 80% of all 1500bp fragments containing factor X can be detected with a 300bp PCR diagnostic. Thus,
as a percentage of the maximum detectable, we can detect 66% (53x100/80) of all possible binding sites +/~700bp from the center of the diagnostic PCR fragment.
Note that the F drops as S decreases, and P, E, or I increase. F = zero when S¡ÂT. Thus, to mazimize the chance of
detecting a binding site, it is best to increase S, and decrease P. Many current ChIP protocols use 500bp sonicaton
fragments. The diagnostic PCR in the above example would miss the binding site in this case (F<0).
*The actual periodicity of cleavage does not affect the calculation. For example, if sonication generates 10bpseparated fragment, S, P, E, and I are all reduced 10-fold, giving the same result as if we assume that sonication
induces breaks at every base. Another assumption is that all regions of the genome are cleaved equally by
sonication, which is probably not the case.
Myles Brown’s lab studies estrogen responsive genes in MCF-7 cells and then treat
cells with -estadiol to get an induced response
Preparing the Estradiol
-take 1mg or -estradiol and add to 1mL of absolute ethanol swirl and add to 50 mL of
media, stock is now 20 ug/mL.
-aliquot and store frozen avoiding freeze thawing
-according to DiRenoza et als they used 100nM of -estradiol to treat MCF-7 cells.
we have 20 ug/mL stock of -estradiol and the molecular weight of -estradiol is 272.4
g/mol
we want then:
=.0000001mol/L*272.4g/mol
=0.00002724g/L of B-estradiol(or 0.02724 ug/mL) in our media
so if I were to make up 50 mL of 17B-Estradiol containing media then I would
or 0.02724 ug/mL * 50mL = 1.36 ug
stock is 20 ug/mL or 0.02 ug/uL
1.36 ug / 0.02ug/uL)=68 ul
add 68 uL of stock to 50 mL of media to get 100nM
or 1.36 ul per mL of media
Chip Protocol modified from Miles Browns lab(with tips from Direnzo and Shang)
Less verbose working copy
1- Cross-link for 10 min at room temp with 1% formaldehyde
2. Rinse cells twice with ice cold PBS and collect in 1 mL ice cold PBS
3. Spin down and wash once with ice cold PBS
4. Cells were then resuspended in 900ul of lysis buffer and incubate 10 min on ice
followed by sonication 3X for 15 s each at maximum setting(want to generate fragments
of about 1500 base pairs- not 500 as in other protocols).
5. Centrifuge at max speed for 10 min at 4 C.
6. Collect the supernatant in a new tube (be sure to set aside a fraction of chromatin for
input control) and dilute in IP dilution buffer 1:10.
7. Pre-clear with 2ug of salmon sperm DNA and protein A-Sepharose(45 uL of 50%
slurry in 10 mM Tris-HCl(pH 8.1) 1mM EDTA) for 2hr at 4ºC. Do a quick spin and
collect the supernatant.
8. Immunoprecipitate 6hr or overnight at 4ºC
9. After immunoprecipitation spin at 4ºC for 10 minutes and transfer supernatant to a
new tube. Now add 45 uL(50% slurry) of protein A-Sepharose and 2 ug of salmon sperm
DNA were added and the incubation continued for 1 hr
10. Precipitates were washed sequentially for ten minutes in
(a) 1X TSEI
(b) 4X TSEII (this wash is important to get rid of background/ non specific DNA)
(c) 1X Buffer III
11. The precipitates were then washed 3X with TE( this wash important to reduce
background)
12. Then samples are extracted two times with 250 uL extraction solution
13. Eluates were combined and heated at 65ºC for at least 6 hr to reverse cross links
14. Samples were purified in QIAquick Spin PCR Purification Kit.
15. 1-5 ul of 50 ul was used to PCR amplify
Solutions:
Lysis Buffer
500 mL
1% SDS
10mM EDTA
50mM Tris-HCl(pH to 8.1)
add protease inhibitors(fresh)
50mL of 10% SDS
5 mL of 1 M EDTA
25 mL of Tris-HCl (pH 8.1)
IP dilution buffer
500 mL
1% tritonX-100
2mM EDTA
150mM NaCl
20 mM Tris-HCl(pH to 8.1)
5mL of 100% Triton
1mL of 1 M EDTA
15 mL of 5 M NaCl
10 mL of 1M Tris-HCl(pH to 8.1)
TSE I
0.1 % SDS
1% TritonX-100
2mM EDTA
20mM Tris-HCl(pH 8.1)
150mM NaCl
500 mL
5 mL of 10% SDS
5mL of 100% Triton
1mL of 1 M EDTA
10 mL of 1M Tris-HCl(pH to 8.1)
15 mL of 5 M NaCl
TSE II
0.1 % SDS
1% TritonX-100
2mM EDTA
20mM Tris-HCl(pH 8.1)
500mM NaCl
Buffer III
0.25 M LiCl
1% NP-40
1% deoxycholate
1mM EDTA
10mM Tris-HCl(pH 8.1)
Extraction Solution
1% SDS
0.1M NaHCO3
Protease Inhibitors
500 mL
125 mL of 1 M LiCl
50 mL of 10% NP40
50 mL of 1 % deoxycholate
500 uL of 1M EDTA
5 mL of 1M Tris-HCl(pH to 8.1)
100 mM PMSF in ethanol, use at 1:100
10 mg per ml aprotinin in 0.01 M HEPES pH 8.0, use at 1:1,000
10 mg per ml leupeptin in water, use at 1:1,000