CHROMATIN IMMUNOPRECIPITATION (CHIP) PROTOCOL FOR YEAST

CHROMATIN IMMUNOPRECIPITATION (CHIP) PROTOCOL FOR YEAST

2 Days Before--Start 5 mL overnight from single colony.

1 Day Before--Subculture overnight into110 mL (large flask for aeration). Dilute so
~2x107cells/mL at convenient time on Day 1.

Reserve Sorvall centrifuge (SS34, 4°C). Cool large rotor (TMA-3E) for TOMY centrifuge.

Day 1
Crosslinking Cells

1. When culture is ready (usually I aim for OD₆₀₀ = 1.2; I've used up to 1.5), add formaldehyde directly to the medium--1% final concentration. Calculation: VOL of culture/36 = mL 37% H₂ C=O. Fix at room temperature with gentle swirling (at least occasionally). Fixation time from 10 min to 2 hours
2. Transfer cells to 50 mL conical tubes and spin down in clinical centrifuge.
3. Spheroplast cells. Volumes are for 100 mL of cells.
4. Resuspend cells in a total of 5 mL 0.1 M TRIS (pH 9.4), 10 mM DTT (freshly prepared), pool, and transfer to Nalgene Oak Ridge tube. Place on ice for 15-20 minutes.
5. Spin down in Sorvall--5 K, 5 minutes, 4°C. Drain.
6. Resuspend cells in 5 mL HEPES/sorb to wash. Spin down (5 K, S minutes, 4°C). Drain.
7. Resuspend in 5 mL HEPES/sorb with 0.5 mM PMSF. Add 30-40 mL 1 mg/mL oxalyticase (can also use 2 mg zymolyase here, as preferred). Incubate at 30°C for 30 minutes with gentle agitation
8. Add l0 mL PIPES/sorb with 0.5 mM PMSF. Spin down immediately--5 K, 5 minutes, 4°C. Drain.
9. Wash spheroplasts (3x). All manipulations on ice.
10. Gently resuspend cell pellet in 5 mL cold PBS with 0.5 mM PMSF. Spin down @ 5K, 5 minutes, 4°C. Drain.
11. Gently resuspend cell pellet in 5 mL cold Triton X/HEPES with 0.5 mM PMSF, 0.8 ug/mL pepstatin A, and 0.6 ug/mL leupeptin. Spin down @ 7 K, 7 minutes, 4°C. Drain.
12. Gently resuspend cell pellet in 5 mL cold NaCl/HEPES with 0.5 mM PMSF, 0.8 ug/mL pepstatin A, and 0.6 ug/mL leupeptin. Spin down @ 7 K, 7 minutes, 4°C. Drain.
13. Sonicate spheroplasts
14. Resuspend cell pellet in 1 mL SDS lysis buffer with 1 mM PMSF, 0.8 ug/mL pepstatin A, and 0.6 ug/mL leupeptin. Transfer to 2 Eppendorf tubes (divide equally).
15. Sonicate suspension on ice for 10 second intervals, with at least 5 minutes on ice in between, until
    DNA is 100-2000 bp range (avg 400-500, Branson Sonifier 250: constant output @ 15-20% power, 6 pulses, 10 sec each.
16. Spin in microfuge at maximum speed, 10 minutes, 4°C-10°C (TOMY @ 15K ~18,000 x gmax)
17. Chromatin solution
18. Transfer sup (maybe cloudy from the SDS) to 15 mL Falcon snapcap tube on ice (expect ~1.1mL).
19. Add 10 mL IP Dilution Buffer with 1 mM PMSF, 0.8 ug/mL pepstatin A, and 0.6 ug/mL leupeptin (10 mL ~9 volumes SDS lysate; final [SDS] ~ 0.1%). Let sit on ice awhile.
20. Spin in TOMY @ 10K (~8,400 x gmax), 10 minutes, 4°C-10°C. Decant sup into 15 mL conical tube. Place on ice. This is the chromatin solution.
21. Set up immunoprecipitations
22. Aliquot chromatin solution to Eppendorf tube (I use 1.5 mL per IP; ~15-20 O.D.₆₀₀ equivalents).
23. Add appropriate volume of primary antibody. Incubate overnight 4°C on Nutator. I usually set up duplicate IPs.
    Note: Always do NO Ab control. Also, if adding competitor antigen, do so several minutes before adding Ab. It may be desirable to denature antigen in SDS first. In this case keep track of the additional SDS and supplement companion IPs to the same final [SDS].

Day 2
Harvest immune-complexes

1. Add 2 ug lambda DNA (previously sonicated to 100-2000 bp size range).
2. Add 40 uL of Protein A Sepharose beads (prepared as 50% slurry in TE/0.1 % BSA/0.1% azide).
3. Incubate 1-2 hr. @ room temp. on Nutator.
4. Wash ips
5. Spin down beads (15K, 1 min. 20°C). Remove aliquots of IP sups (< 0.5 mL), if desirable. Aspirate remaining sup.
6. Wash beads sequentially with 1 mL of each of the following buffers, nutating beads 3-5 minutes in each buffer:
   TSE-150
   TSE-150 or TSE-500
   LiCl/Det
   TE
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Day 3
Recover Precipitated Chromatin

1. Spin down EtOH ppts.
2. Wash with 70% EtOH. Dry briefly in speed vac. Resuspend in 100 uL TE. Let sit on ice a while. Totals and Sups will be lumpy and gunky--break up with pipet tip. Be careful not to lose material on the tip.
3. Proteolytic Digestion of Chromatin
4. Add 25 uL 5x Proteinase K Buffer, mix, then add 1.5 uL Proteinase K solution (Boehringer Mannheim).
5. Incubate at 42°C for 1-2 hr. Aggregates in Totals and Sups should disappear.
6. Add 175 uL TE to Pellet samples (fimal vol = 300 mL) and 275 uL TE to Totals and Sups (final vol = 400 uL).
7. Organic extractions and recovery of DNA
8. Make fresh phenol:chloroform:isoamyl alcohol (25:24:1; PCI).
9. Extract Pellets once with 300 uL PCI, then once with 300 mL CHCl₃ . No need to back extract, provided only a few uL aqueous left behind.
10. Extract Totals and Sups twice with 400 uL PCI and twice with CHCl₃ . Back extract organic phases with an additional 100 uL TE. Combine with primary aqueous phase.
11. Add glycogen (2-5 mg) to Pellets. Add 1/10 volume 3 M NaOAc and 2 volumes absolute EtOH to all samples. Precipitate overnight at -20°C.
    

Day 4
Recovery of DNA

1. Spin down EtOH precipitates. Wash with 70% EtOH. Dry briefly in speed vac.
2. Resuspend Pellets in 150 uL TE (equivalent to 10 uL chromatin solution per 1 uL).
3. Resuspend Totals and Sups in TE volume equivalent to 1 mL chromatin solution per 1 uL (e.g. 500 uL TE for Total corresponding to 0.5 uL chromatin solution).

PCR
I use 3 mL of each sample to program a 50 uL PCR reaction. (Note: Given the volumes used to resuspend various samples, the chromatin solution equivalent for Total or Sup is 1/10 that used for Pellet). Controls include no DNA and plasmid or good genomic DNA as a positive control.

Reaction Conditions:	DNA (3 uL)	Program: 95°C, 3 minutes
(in 50 pL)	lx Taq Bfr
	1.5 mM MgCl2	95°C, 30 seconds
	0.6 uM each primer	Tm -5°C, 45 seconds
	0.2 mM dNTPs	72°C, 60 seconds
	0.5 uL Taq	Amplify for 24 cycles, total.
		72°C, 5 minutes
		4°C

Add 5x Bluejuice. Analyze l/3 to 1/2 reaction on 2.5% agarose gel or 8% PAGE. Remember to be quantitative!! Photograph gel with 55 film and scan in negative to "quantitate."

Slot Blot
Dilute aliquot of each sample in 6xSSC. Denature at 100°C ~10 minutes. Place immediately on ice. Apply samples to Nytran. Wash 2x with 6xSSC. UV crossTink filter prior to hybridization.

Southern Blot
Usually need to digest at least 1/2 of Pellet samples to see a signal; therefore, aliquot 75 mL to new Eppendorf tube and reduce volume in speed vac. Alternatively, if Southern blot analysis is anticipated, resuspend Pellets in a smaller volume initially. For Totals and Sups, digest an amount of chromatin solution equivalent to 1/5 to 1/10 amount used for Pellets.

For CEN3, AluI is the diagnostic digest. Digest for several hours to overnight. Add RNase to bluejuice for Totals and Sups prior to loading onto gel. Run a 2.5% agarose gel. Transfer to GeneScreen. (I leave out the HCl treatment when preparing gels for transfer.). UV crosslink prior to hybridization.

Reagents Needed for ChIP

37% formaldehyde stock solution

0.1 M DTT stock

1 M TRIS, pH 9.4 stock

1 mg/mL Oxalyticase stock or 10 mg/mL Zymolyase stock

PMSF, pepstatin A, leupeptin stocks

HEPES/sorb 250 mL:
20 mM HEPES, pH 7.4 5 mL 1 M HEPES
1.2 M sorbitol 150 ml, 2 M sorbitol

PIPES/sorb 250 mL:
20 mM PIPES, pH 6.8 5 mL 1 M PIPES
1 mM MgC12 0.25 mL 1 M MgC12
1.2 M sorbitol 150 mL 2 M sorbitol

PBS

Triton/HEPES Wash 250 mL:
0.25% Triton X-100 3.13 mL 20% Triton X-100
10 mM EDTA 5 mL 0.5 M EDTA
0.5 mM EGTA 0.5 mL 0.25 M EGTA
10 mM HEPES, pH 6.5 2.5 mL 1 M HEPES

NaCl/HEPES Wash 250 mL:
200 mM NaCl 10 mL 5 M NaCI
1 mM EDTA 0.5 mL 0.5 M EDTA
0.5 mM EGTA 0.5 mL 0.25 M EGTA
10 mM HEPES, pH 6.5 2.5 mL1 M HEPES

SDS Lysis Buffer 100 mL:
1% SDS 10 mL 10% SDS (UltraPure)
10 mM EDTA 2 mL 0.5 M EDTA
50 mM TRIS, pH 8.1 5 mL 1 M TRIS

IP Dilution Buffer (for 1:9 dilution) 250 mL:
0.01% SDS 0.25 mL 10% SDS
1.1% Tnton X 100 13.8 mL 20% Triton X 100
1.2 mM EDTA 0.6 mL 0.5 M EDTA
16.7 mM TRIS, pH 8.1 4.2 mL 1 M TRIS, 8.1
167 mM NaCL 8.35 mL 5 M NaCl


Sonicated lambda DNA. Store at 4°C with 0.1% azide.

Protein A Sepharose Bead Buffer 50 mL:
50 mL TE
2.5 mL 2% BSA (=166mL of 30% stock)
0.5 mL 10 % azide (=1.54M azide_
(or 0.77mL of IMaside [770 mL]

TSE-150 Wash (= IP conditions) 250 mL:
0.1% SDS 2.5 mL 10% SDS
1% Triton X-100 12.5 mL 20% Triton
2 mM EDTA 1.0 mL 0.5 M EDTA
20 mM TRIS-HCl, pH 8.1 5.0 mL 1 M TRIS, 8.1
150 mM NaCl 7.5 mL 5 M NaCl

TSE-500 Wash (Optional) 250 mL:
0.1% SDS 2.5 mL 10% SDS
1% Triton X-100 12.5 mL 20% Triton
2 mM EDTA 1.0 mL 0.5 M EDTA
20 mM TRIS-HC1, pH 8.1 5.0 mL 1 M TRIS, 8.1
500 mM NaCl 25 mL 5 M NaCl

LiCl/Detergent Wash 250 mL:
0.25 M LiCl 2.65 g LiCl (42.39)
1% NP-40 25 mL 10% NP-40 ?
1% DOC 50 mL 5% DOC ?
1 mM EDTA 0.5 mL 0.5 M EDTA
10 mM TRIS-HCl, pH 8.1 2.5 mL 1 M TRIS, 8.1

TE, pH 8.0

1% SDS/0.1 M NaHCO3 50 mL:
5 mL 10% SDS
5 mL 1M NaHCO3 ?

5x Proteinase K Buffer 10 mL:
50 mM TRIS, pH 0.5 mL 1 M TRIS
25 mM EDTA 0.5 mL 0.5 M EDTA
1.25 % SDS 1.25 mL 10 % SDS

Proteinase K Solution (Boehringer Mannheim, 1413 783), ~18.6 mg/ mL

Glycogen stock