Yeast Chromatin Immunoprecipitation (ChIP)

Hahn Lab

July 2003

Chromatin IP Method

Day 1

1.Start 5 ml o/n cultures of strains to be tested

Day 2

1.Inoculate 50 ml of the desired media with a volume of a saturated o/n culture and grow o/n with shaking at 200 rpm.The volume of cells inoculated should be such that the culture reaches the desired A600 the next morning.(For example,130 microliters of a wild-type strain/100 ml YPD inoculated at 5:00 pm will be at an A600 of ~1.2 at 8:00 am next morning)

Day 3

1.Check A600 until desired cell density is reached (usually ~ 1.5)

2.Once the desired cell density is reached remove the culture from the shaker

3.To the 50 ml culture add 1.39 ml formaldehyde (1% final),mix by swirling

4.Incubate at room temperature 10-20 min with occasional swirling (Times ranging from 1min to hours for crosslinking ChIP samples have been used in the literature.It is important to treat all cultures with formaldehyde for the same length of time.)

5.Add 2.75 ml 2.5 M glycine (125 mM final),mix by swirling

6.Incubate for 5 min at room temperature

7.Pellet cells in GSA at 5000 rpm for 10 min,decant supernatant

8.Wash cells with 20 ml cold TBS+125 mM glycine

9.Wash cells with 20 ml cold TBS,decant supernatant

10.Resuspend cells in the TBS that remains in the bottle after decanting (add 0.5ml TBS if necessary),and transfer the cells to a Marsh 2.0 ml tube

11.Pellet cells in a cold microcentrifuge for 10-30 sec,remove all supernatant

12.Freeze cell pellet on dry ice and store at 70 degrees C

Days 3 and 4 are easily combined if the cells are ready early in the day.

Day 4

1.Resuspend cell pellets in 400μl ChIP lysis buffer+protease inhibitors

2.To each sample add an equal volume (about 0.5 ml)of glass beads (425-600 micron diameter)

3.Lock every Marsh tube to ensure the tubes don’t open during lysis

4.Lyse cells in the Breeden Lab FastPrep vortex 3 x 40 sec,with 40 sec pauses between runs,at level 4.5 M/s (A2-124 cold room)

5.Remove locks and pierce tube bottoms with a hot 20 G 1.5 needle (yellow)

6.Put pierced tubes into 15 ml Sarstedt tubes and spin in a cold clinical centrifuge at top speed for 1 min (the extract will transfer to the 15 ml tube leaving the glass beads in the 2ml tube which can then be discarded)

7.Emulsify the insoluble pellets with their supernatants and transfer to 2.0 ml round bottom tubes (the chromatin is primarily in the insoluble fraction)

8.Sonicate extracts in Breeden Lab water bath sonicator at level 5,100% duty,4 x 30 sec with 30 sec pauses on ice between runs (sonicator output should be approximately 20-25 when sonicating samples)

9.Clarify extracts in cold room microcentrifuge for 10 min at top speed

10.Transfer supernatants to 1.5 ml tubes and centrifuge again in cold room for 5-10 min at top speed

11.Transfer supernatants to 1.5 ml tubes

12.Quantitate extracts by Bradford assay: 1μl of a 1:6 dilution works well in the assay for samples taken from cultures between OD1 and 2

13.Freeze extracts on dry ice,and store at -70 degrees C

Day 5

1.Thaw extracts on ice and transfer a volume of extract (1mg total protein)to a new 1.5 ml tube for IP

2.Include necessary controls (i.e.no Ab control; untagged strain+ Ab)

3.Bring all 1mg aliquots to 200μl with ChIP lysis buffer+protease inhibitors,mix well (thorough mixing is very important)

4.Transfer 2μl from each 200μl IP sample to a new 1.5 ml tube with 150μl ChIP elution buffer and set aside until the IP’s are complete (these are the INPUT samples)

5.Add Ab to each IP sample except for the no-Ab control,nutate in cold room for 3 hrs

6.When there is 30 min left from step 5 prepare rProtein A (or G)Sepharose beads

Spin down beads and remove supernatant

Wash beads twice with 1ml ChIP lysis buffer+protease inhibitors per 250μl bead volume

Resuspend beads after washing in an equal volume of ChIP lysis buffer+protease inhibitors to make 50% slurry (prepare 25% more beads than you calculate to be necessary)

7.Add 60μl of 50% bead slurry to each IP sample,nutate in cold room 1hr

8.Spin beads down in cold room microcentrifuge for 10-15 sec,remove the supernatant,and wash with the following solutions

2x 1ml ChIP lysis buffer+protease inhibitors

2x 1ml ChIP high salt lysis buffer+protease inhibitors

2x 1ml ChIP wash buffer

2x 1ml 1xTE (8.0)

9.After the last wash draw off as much liquid as possible from the beads using a finely pulled Pasteur pipette

10.Resuspend the beads in 85μl ChIP elution buffer and incubate at 65 degrees C and 950 rpm in a Thermomixer for 10 min

11.Spin down beads in microcentrifuge and transfer 75μl of the supernatant to a new 1.5ml tube

12.Add 75μl to the beads,repeat steps 10 and 11,and combine the two 75μl elutions (IP)

13.For each extract being tested you should now have an IP sample (150μl)and an IN sample (150μl)plus any controls

14.Incubate all IP and IN samples at 65 degrees C overnight to reverse crosslinks

Day 6

1.Add 750μl Qiagen buffer PB to each IP and IN sample

2.Purify using the Qiagen PCR purification kit,elute DNA in 50μl buffer EB

3.Analyze the samples by quantitative PCR using an appropriate protocol

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ChIP Buffers

2.5 M glycine (filter sterilize,store at room temperature)

1x TBS⁺ 125 mM glycine (store at 4 degrees C)

1x TBS (store at 4 degrees C)

ChIP lysis buffer (filter sterilize,store at 4 degrees C)

50 mM HEPES pH 7.5

140 mM NaCl

1% TritonX-100

0.1% Sodium Deoxycholate

1mM EDTA

ChIP lysis buffer (High Salt)(filter sterilize,store at 4 degrees C)

50 mM HEPES pH 7.5

500 mM NaCl

1% TritonX-100

0.1% Sodium Deoxycholate

1 mM EDTA

ChIP wash buffer (filter sterilize,store at 4 degrees C)

10 mM Tris pH 8.0

250 mmliCl

0.5% NP-40

0.5% Sodium Deoxycholate

1 mM EDTA

1x TE pH 8.0 (filter sterilize,store at 4 degrees C)

ChIP elution buffer (store at room temperature)

50 mM Tris pH 8.0

1% SDS

10 mM EDTA

Protease inhibitors

PMSF,Benzamidine,Pepstatin,Leupeptin,and Chymostatin

Phosphatase inhibitors (store at �20 degrees C)

Solution A (100x)for 10 ml

100 mM sodium pyrophosphate 0.45g

100 mM sodium orthovanadate 0.18g

Water to 10 ml

Solution B (100x)for 10ml

100 mM beta-glycerophosphate 0.29g

100 mM EGTA 0.38g

1M NaF 0.42g

Water to 10 ml

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Notes on ChIP method:

Day 2

Step 1.A dilution of 1:1000 of a saturated culture of SHy278 will grow to approximately OD₆₀₀ 1.0 in 16 hours when grown at 30 degrees C.

Day 3

Step 4.The duration of formaldehyde treatment is a point of optimization.Shorter treatments will generally yield higher concentrations of soluble protein,but less extensive cross-linking of proteins to DNA.Longer treatments will yield the opposite,and consequently there will be less material in the extracts for doing IP’s.However,this is not the first step that should be optimized.20 minutes is probably a good starting point for this step unless the effect being observed is expected to occur in a very short time frame.In that case 5-10 minutes formaldehyde treatment may be more appropriate.Optimization of this step involves maximizing the IP/IN ratio for the protein-DNA interaction being assayed,and keeping the duration of formaldehyde treatment short.This can be examined by PCR after IP conditions have been optimized by Western analysis.

Day 4

Step 1.If assaying for phosphorylated proteins,add phosphatase inhibitors at this and subsequent steps when protease inhibitors are added.

Step 4.Pliers are often useful for loosening the knobs on the FasePrep vortex when removing the lysates.

Step 8.The water bath sonicator develops a film over the membrane used to sonicate the samples.For best results run the sonicator at level 10 for a few seconds to break the film up before adding samples to the water and sonicating at level 5.It may also be a good idea to add some ice to the water bath to keep the samples as cool as possible.

Quality Control: After the extracts are made you can verify efficient shearing of DNA by the following:

1.mix 20μl extract⁺ 1μl 10% SDS⁺ 0.5μl proteinaseK

2.incubate 1hr at 37 degrees C followed by 2 hrs at 65 degrees C

3.phenol/chloroform extract the samples

4.chloroform extract the samples

5.EtOH ppt DNA with 10μg/ml glycogen (final concentration)

6.resuspend DNA in 20μl TE

7.treat with 40μg/ml RNAse for 1hr at 37 degrees C

8.run half of each sample on a 1.5% agarose gel with EtBr with an appropriate DNA sizing ladder (sheared DNA should run between 100 bp and 1000 bp)

Step 12.ChIP extracts made as described are generally 15-25 mg/ml

Day 5

Step 5.The amount of Ab used in the assay must be determined empirically.Important considerations include: the fraction of your protein-of-interest the Ab is able to pull down,how much the protein is left after elution,and the ratio of specific PCR signal to noise.Before proceeding to the main experiment it is important to optimize the IP conditions using a combination of Western and PCR analyses to address the aforementioned issues.

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ChIP PCR using ³²P-dCTP

1.Dilute purified DNA samples in water

a.Correct dilutions will need to be determined empirically by titration.Reactions should be set up such that the signal for IP and INPUT samples are approximately equal.

b.Several dilutions of each sample being assayed should be made,and 10μl of each dilution used in PCR.Using several dilutions of each sample allows confirmation that the reactions are in the linear range.

2.Make a 2x PCR mix for every set of primers being used.For every 20μl reaction add to the mix the following:

a.2μl 10x Mg free buffer

b.2μl 2 mM dNTP mix

c.0.6μl 50 mM MgCl₂

d.1μl each 20 uM primer

e.2.8μl water (if 4 primers are being used together add 0.8μl water/rxn)

f.0.4μl Platinum Taq

g.0.2μl ³²P-dCTP

3.Add 10μl PCR mix to each 10μl DNA sample

4.Amplify DNA using the following program:

a.95 degrees 2 minutes

b.95 degrees 30 seconds

c.anneal 30 seconds

d.72 degrees 1 minute

e.repeat steps b-d 19-25 more times (the number of cycles must be optimized empirically)

f.72 degrees 3 minutes

g.hold at 4 degrees

5.Spin down reactions and add 4μl 6x loading dye to each

6.Run 10μl of each reaction on 6% PAGE

a.use 0.5x TBE as the running buffer

b.run the gel at 180 volts (constant)for about 75 minutes

c.once the bromophenyl blue is 2/3 to 3/4 down the gel stop running

d.some ³²P-dCTP will reach the lower buffer,but most will remain in the gel below the BPB if it is not run too long

e.cut the gel off just above the BPB and throw the bottom section of gel into the radioactive DSW bin

f.dry the gel on two pieces of Whatman 3 MM paper