Chromatin Immunoprecipitation (ChIP) Protocol
From Brehon Laurent's lab with Koshland lab changes

DAY 0
0. Grow cells overnight in appropriate media.

DAY 1
1. When cells reach OD600 ~0.5, add NZ/HU etc. After arrest, fix 100ml of cells by shaking in 1% formaldehyde [2 x 0.7 ml of 37% Formaldehyde] for 15 min @ RT. I use ~50-100 OD equivalents, and fix 2hours at RT for cohesin subunits, Also mif2 signal increases with increased fixation time (2h works the best). I only fix for 15 minutes for Smc3-HA tagged protein. Also, for A364A background, the 50 ODs seems to work better than 100 OD, they don't seem to break open as easily as JKM background if there are more cells.
2. Collect cells by spinning 2500rpm, 4°C, 2min.
4. Wash 2X with 10ml cold 1X TBS (or 1X PBS). Spin 2500rpm, 4°C, 2min.
5. Quick-freeze cells in dry ice for 5 min before transferring to -80°C freezer.

DAY 2
6. Thaw cells on ice for >30min.
7. Wash cells with 10ml FA buffer (1mM PMSF 1:200, 1mM DTT 1:1000). Spin 2500rpm, 4°C, 2min.
8. Resuspend in leftover FA buffer, transfer to 1.5ml Eppendorf tube.
9. Pellet cells by spinning 6000rpm, 1min, 4°C.
10. Prepare FA buffer containing:

1.0mM DTT (1:1000)
2.0mM PMSF (1:200)
1 protease mini tablet/10ml of buffer

11. Resuspend cells in 0.4ml FA buffer. Add 0.6ml pre-chilled glassbeads.
12. Break cells at room temperature using a TOMY tube mixer (1min x10, rest for 1min in ice-water) (or in cold room, for 25 min (5, 10, 10 separately), rest in ice-water for 2 min in-between). For A364A background, I actually do 30 minutes (10, 10, 10 respectively) because they seem a bit harder to lyse than the JKM background.
13. Collect cell lysate by tube puncturation on bottom of Eppendorf tube, placing in 15ml conical with receiving, clean Eppendorf inside, and spinning 2000rpm, 2min, 4°C. Pipet cell lysate up/down and transfer to clean tube.
14. Sonicate ( 3/16” tapered microtip )


Cell lysate is first made up to 1.2ml with FA buffer (with freshly added protease inhibitors) and transferred carefully to a 2ml round eppendorf tube, Sarstedt #72.695)

Sonication - breaking DNA into 500 bp fragments. Set the sonicator so that the output is between 20 and 40 and duty cycle = constant.
Repeat 6 times for each tube:
1. Rinse the sonifier with water, then ethanol and dry with kimwipe.
2. Sonicate each sample for 10 sec
3. Let samples sit on ice for 2-3 minutes between sonication

13. Clear chromatin extract by centrifugation @ 4°C and 14,000rpm for 5 min, then transfer supernatant to a new Eppendorf tube and spin again for 25 min.
14. Transfer supernatant to a new Eppendorf tube.
15. I bring the volume to 4.5ml by adding FA buffer with protease inhibitors, and it is enough to set up 4 different IPs (1 ml/IP) and for 300ºl Total/Input chromatin )
16. Incubate in 1.0ml FA buffer (w/ proteinase inhibitors), appropriate amount of chromatin with antibody @ 4°C overnight on Nutator. Set aside 300ºl Total/Input chromatin. This can be put in the -20 overnight. For different antibodies, I use 1.5ºl of Mcd1 or Pds5 antibodies from Vinny/ IP. I use 5ul of the HA antibody.

DAY 3
18. Add 60ºl of 50% (v/v) protein A slurry and incubate for an additional 2-3 hr at 4°C.
19. Centrifuge @ 2,000rpm for 1min
20. Wash beads (using round long tip to remove supernatant) on nutator @ RT x 4min with:
1 ml FA buffer (w/ 1mM PMSF 1:200), once
1 ml FA buffer (w/ 500mM NaCl 1:10, 1mM PMSF 1:200), once
1 ml LiCl-detergent, once
1 ml 1X TE, twice (leaving as little volume as possible)
21. Add 300ul bead elution buffer, mix by inverting 5X and spinning 6000rpm, 1min.
22. Incubate on nutator @ RT for 20 min.
23. Spin 6000rpm, 3 min @ RT.
24. Using a long flat tip, transfer all supernatant to a new tube.
25. Reverse cross-linking of both totals and IP'd samples.
(ChIP) Add 15ul 5M NaCl, incubate @ 65°C for 4-6hr.
(total) Add 2.5ul 5M NaCl, incubate @ 65°C for 4-6hr.
26. Freeze samples overnight at -20°C.

DAY 4
27. (ChIP) Add 1/50 vol 0.5M EDTA 315/50 = 6.3ul
1/25 vol 1.0M Tris-HCl, pH 6.8 315/25 = 12.6ul
20ug proteinase K (20mg/ml) 1ul
(Total) Add 1/10 vol 10% SDS 302.5/10 = 30.25ul
1/50 vol 0.5M EDTA 302.5/50 = 6.05ul
1/10 vol 1.0M Tris-HCl, pH 6.8 302.5/10 = 30.25ul
40ug proteinase K (20mg/ml) 2ul
Incubate @ 42°C for 2 hr.
28. Extract @ RT with phenol:chloroform:isoamyl alcohol (25:24:1) once for IP's and twice Totals and with chloroform once.
[ChIP: use 300ul; totals: use 400ul]
Centrifuge at 25°C, 13,000rpm, 5min.
29. Add: 20ug glycogen (1ul, from Roche) ChIP Totals
1/10 vol 3.0M NaOAc, pH 5.2 30ul 40ul
2 vol EtOH 600ul 800ul
Precipitate DNA @ -20°C (~8 hrs to O/N precipitation)
30. Spin @ 13,000rpm for 25min (@ 10°C). Wash precipitate with 1ml 70% EtOH. Spin 13,000rpm, 5min, 10°C. Air dry. I only air dry for about 10 minutes. Otherwise it gets hard to resuspend.
31. Resuspend DNA in 200ul (or 400ul, depending on the yield) TE. I usually resuspened in 200ul and then do a few test pcrs, and if the signal is really bright, dilute my samples to 400ul.
32. Allow DNA to completely dissolve in TE at least overnight @ 4°C. Sometimes I give it a few days in the fridge, or leave it at room temp for a while. You really want to make sure that your DNA is resuspended, especially if you let them dry too long before adding the TE.
33. Analyze DNA presence by PCR. Dilute totals 1:30 and use 5ul for PCR. This creates a 100X difference between Chip and totals. For IP's use 5 ul of undiluted samples.

PCR conditions 95C, 3min
95C, 30sec
57C, 45sec
72C, 60sec
72C, 5min
4C

Setting up your PCR:
Take your 100uM primers (Forward and Reverse) and make a primer stock master mix which contains both primers. Add 25ul of each primer to 250ul of TE.

PCR reaction:
Template 5ul
5x buffer(containing loading dye) 3ul
MgCl2 2 ul
dNTP .3 ul
taq .1 ul
diluted primer stock 1.8ul
dH20 2.8 ul

Make a master mix containing everything except primer and template DNA. Add primers first to the bottom of the tube on ice. Then add template to the side of the tube. Finally add master mix. Add caps and mix by a slight vortex. Spin down in a centrifuge to mix and run PCR. For IPs were the signal is low (you can always dilute your totals further and run for an additional 2 cycles).

Run pcrs on a gel (I usually load 11-13ul). 200 ml gel using 2.5% NuSieve Agarose containing 30ug of EtBR, run at 120V for 50 minutes. Do not add EtBr to your sample buffer! Take a picture and quantitate with IP labs software.


Solutions:
Formaldehyde: 37%, Fisher

5X FA Buffer
FA: 50mM HEPES, pH 7.5 250mM HEPES, pH 7.5 12.5ml 1M HEPES
150mM NaCl 750mM NaCl 7.5ml 5M NaCl
1mM EDTA 5mM EDTA 500ul 0.5M EDTA
1% Triton X-100 20X ad 50ml H2O
0.1% DOC (freshly-made) 100X
0.1% SDS 100X



LiCl-detergent:

0.25M LiCl
1% NP-40
1% DOC
1mM EDTA
10mM Tris-HCl, pH 8.1

Bead Elution Buffer:
1% SDS/0.1M NaHCO3



TE: 10mM Tris-HCl, pH 8.0
1mM EDTA

Proteinase K: 20mg/ml in H2O

Glycogen: 20mg/ml

PMSF: 200mM in EtOH, make fresh each time

1. Aspirate after final wash (very carfully, get as close to beads as possible without loosing any)
2. Add 250 µl 1% SDS/0.1 M NaHCO3.
3. Incubate &Mac179;20 minutes at room temperature on Nutator.
Pam's note: OK to incubate longer or stop here for a while
4. Spin @10 K, 1 min, RT.
5. Transfer supernatant to new tube using gel loading pipet tips, making sure no beads are transferred. Be patient and let the liquid drain from the pipet tip.
6. Repeat steps 2-5.
7. Transfer supernatant to same new tube, using the method in step 6, but when most of it is transferred, get the last bit by pressing the tip against the bottom of the tube while pushing some air out (so no beads enter) and pipet up as much of the remaining liquid as possible.
8. Add 20 µl 5M NaCl.
9. Vortex. Spin down briefly. Make sure lids are closed completely.
10. Heat at 67&Mac251;C for 6 hours to overnight to reverse the formaldehyde crosslinks.

o Totals

1. Aliquot 300µl amounts of Chromatin Solution (no antibodies).
2. Add 3.2µl 5M NaCl.
3. Vortex.
4. Heat at 67&Mac251;C from 6 hours to overnight to reverse crosslinks.
Dave's Note: I usually prepare 2 total samples. That way, if something goes wrong during extractions, you don't have to start over from the beginning.

Pam's note: Might want to use lid locks or put heavy weight on top of all tubes to prevent evaporation.



Day Three

o Proteinase K Treatment

1. To IP's: Add 20µl 1M Tris-HCl pH 6.8 and 10µl 0.5M EDTA (make up cocktail and add 30µl to each tube)
2. To Totals: Add 33.3µl 10% SDS and 5.5µl 0.5M EDTA
3. Vortex.
4. Add 2µl Proteinase K solution.
5. Invert 3-4 times to mix.
6. Incubate @42&Mac251;C for 2 hours.


o Extractions

***For all extractions:
o Mix on TOMY mixer (max speed) for 2 minutes.
o Spin @10K, 5 min, room temperature. ***

1. Make phenol/chloroform/isoamyl alcohol (PCI) (25:24:1). Spin PCI @2K, 5min, RT, to make sure there is a water layer on top. Balance by weight (PCI is denser than water).
2. Extraction of IP's
o Extract once with equal volume PCI (500µl).
o Extract once with equal volume Chloroform (500µl).
o YYH recommends extracting with equal volume Chloroform (500µl) a second time.
3. Extraction of totals:
o Add 60µl TE.
o Extract twice with equal volume PCI (400µl).
o Extract twice with equal volume Chloroform (400µl).
o Back extract organic phases with 100µl TE.

For the totals extraction, set up five rows of tubes. Move the aqueous layer of the first PCI extraction to the second row. While extracting the second row with PCI, back extract the first row with TE. Again, move the aqueous layer from the first row to the second, from the second to the third, then extract the third row with chloroform and the second row with TE. Continue until all four extractions and back extractions are complete.

4. After extractions of both ChIP pellets and totals add:
5µg glycogen (from 20µg/µl stock - 0.25µl per tube)
50µl 3M NaOAc
1ml EtOH
Make a cocktail of glycogen and NaOAc, then add the EtOH separately.
5. Leave @-20&Mac251;C overnight.



Day Four

o Spin down all EtOH precipitates (ChIP and totals) @15K, 20min, 4&Mac251;C.
o Decant immediately after spin stops. Be careful because ChIP pellets should be VERY VERY TINY. If they are big it means there is SDS and they should be re-extracted with CHCl3 one more time and re-precipitated.
o Wash with 500µl 70% EtOH (DNA is not soluble in 70% EtOH).
o Spin @15K, 10min, 4&Mac251;C.
o Decant wash.
o Dry in speedvac.
o Resuspend in TE:

1. For ChIP pellets, we usually use 1µl/10 µl chromatin solution used for IP.
(For MIF2 and CSE4-HA under wild type conditions, can dilute in 0.5 µl/1 µl chromatin solution used for IP.)
amt used _________
2. For Totals, use 1µl/1 µl chromatin solution
amt used _________


Recipes


FA lysis buffer (500ml)

25 ml 1M HEPES-KOH, pH 7.5
14 ml 5M NaCl
1 ml 0.5M EDTA, pH 7.6
25 ml 20% Triton-x-100 (by vol)
10 ml 5% DOC (deoxychlolic acid)
425 ml sterile water

Add the following supplements to 10ml FA lysis buffer, when required:

10 µl 1mg/ml Leupeptin (Leu)
10 µl 1mg/ml Pepstatin (Pep)
50 µl 0.2M PMSF

Vortex immediately after adding PMSF.


FA Wash Buffer (500ml)

25 ml 1M HEPES-KOH, pH 7.5
50 ml 5M NaCl
1 ml 0.5M EDTA, pH 7.6
25 ml 20% Triton-x-100 (by vol)
10 ml 5% DOC (deoxychlolic acid)
389 ml sterile water

1X TE (100ml)

1 ml 1M Tris pH 8
200µl 0.5M EDTA pH 7.6

Fill to 100ml with sterile water.

LiCl/Detergent Wash (250ml)

2.65g LiCl
25 ml 10% NP-40
50 ml 5% DOC
0.5 ml 0.5M EDTA
2.5 ml 1M Tris pH 8.1



Alternative Breakage Buffer (probably less background with anti-HA)
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:
1.1% Triton 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