ChIP Protocol-Mechanical Breakage & FA Lysis Buffer


Day -2:

o Start a 5ml overnight culture from a single colony.

Day -1:

o Start a larger overnight culture using part of the 5ml culture (subculture).

Day One - Part One:

1. Grow culture until it is in log phase: OD600=0.7 - 1.2
2. For each sample you collect, put the appropriate amount of 37% formaldehyde in a 50ml tube and label. The calculation for the correct amount of 37% formaldehyde to use is: volume of culture/36. This is a 1% final concentration of formaldehyde. For 50ml samples, use 1.4ml 37% formaldehyde.
3. When you are ready to fix a sample, fill one of the tubes with culture to the line right under the 50ml marking. Mark the time on the tube and put it on a flat shaker.
4. At 1hr and 50 minutes after the fixing start time:

o Spin tubes @ 2K for 5 min, RT.
o Decant and blot on paper towel.
o Vortex pellet.
o Resuspend pellet in about 25ml of 1X Buffer A (TBS).
o Repeat steps 1 to 4.
o Repeat steps 1 to 3.
o Resuspend in 1ml of 1X Buffer A (TBS).
o Transfer to 1.5ml tube.
o Spin @10K, 1min, RT, aspirate supernatant.
o Put tube with pellet on ice.
o To store the pellets overnight, put tube in dry ice and store at -80&Mac251;C until ready for the next step.

Dave's Notes:
1. This protocol was written assuming 50-ODeq pellets. Volumes can be adjusted as required for the specific application.
2. We use Fisherbrand 50ml disposable, sterile, conical tubes to harvest. It's not important that the harvesting is done exactly like this. What is important is the fixation time.
3. About fixation: the amount of fixation time used depends upon the protein of interest. For histone proteins, short fixation times (10-20 minutes) are required; however, for MIF2, the amount of DNA in the IP increases with increasing fixation time. When testing a new protein, I usually harvest two pellets-one fixed for 20 minutes, the other for 2 hours and compare. Also, TBS (tris-buffered saline) quenches the fixation reaction; therefore, the fixation time is considered the amount of time between the addition of the formaldehyde and the first resuspension of the cells in TBS. This is why we begin the spins early.

Day One - Part Two:

o Thaw pellets on ice for 10-30 min (as needed).

o Bead Break Cells (Mechanical Breakage):

1. Resuspend pellets in 250µl of FA lysis buffer w/ protease inhibitors by pipeting up and down. (NOTE: might want to use 1% SDS Lysis Buffer if having background with anti-HA; see Pam's older protocol & alternative recipe at end for lysis buffer and IP Dilution Buffer)
2. Add acid-washed glass beads, so there is a 1 to 2mm layer of liquid above the beads.
3. Vortex in the Tomy Mixer. Set it to level 10, mix 8 times for 1 minute each, and put on ice for one minute between mixes.
o o o o o o o o
o Spin @ 500rpm for 15 seconds.

o Add 250µl of FA lysis buffer w/ protease inhibitors.

o Stack Transfer - Transfer lysate to new tube

1. Label and open new tubes.
2. Wipe outside of old tube with ethanol.
3. Turn tube upside down and “flick” tube to get all liquid to top of tube.
4. Using a Bunsen burner, heat the tip of an 18-gauge needle until red.
5. Make a hole in the bottom of the tube. The beveled tip of the needle should be half way into the tube.
6. When the plastic has hardened, pull out the needle.
7. Nest the full tube into the empty tube.
8. Spin all of the nested tubes @ 3K, 1 min, 4&Mac251;C. All of the liquid - without beads - should now be in the bottom tube.

o Sonication - breaking DNA into 500 bp fragments. Set the sonifier to power=1.5 and duty cycle = constant.
Dave's Note: we use Branson Sonifier 250 with a 3/16” tapered microtip.

Repeat 6 times for each tube:
1. Rinse the sonifier with water, then ethanol and dry with kimwipe.
2. Sonicate leftover FA lysis buffer w/ supplements for a moment.
3. Put open sample tube in round, cold rack (from freezer) and sonicate for 10 seconds. Avoid aerating the sample.
4. Let sample sit on ice for 5 minutes.
Dave's Note: ice time can be decreased. I usually only wait 1-2 minutes.

o Clarify Sonicate

1. Spin @15K, 20min, 4&Mac251;C (clarifying spin). Transfer supernatant to Falcon snap cap tube, discard pellet.
Dave's note: we use 15ml snap cap, round bottom tubes from Falcon (cat#35-2059)

2. Dilute supernatant 1:9 with ice-cold FA Lysis Buffer w/ Protease Inhibitors.
3. At this point, it's best to let the diluted extract sit on ice for 30 min to allow time for non-specific precipitation of material.
4. Spin in centrifuge @8250 rpm, 10 min, 4&Mac251;C.
5. Transfer supernatant to new 15 ml conical tube - this is the Chromatin Solution.

o Set up ChIP's

1. We do our IP's in microtubes, using about 1 ml of Chromatin Solution per IP.
2. Add antibodies to the appropriate dilution. Remember to include a no antibody control for each yeast strain used. Store remaining Chromatin Solution at 4&Mac251;C; at least 300 µl are needed for preparation of Total DNA content.
3. Nutate the IP's overnight at 4&Mac251;C.



Day Two

o Harvest Immune Complexes

1. To each tube, add:
4 µl sonicated lambda DNA (Gibco BRL cat#25250-010, must be sonicated like the ChIP extracts & check on gel to verify small size-add azide to 0.01%)
40 µl Protein A Sepharose Beads (prepared as 50% slurry in TE/0.1% BSA/0.1% azide)
2. Incubate 2 hr. @ RT on Nutator.

Pam's note: If using monoclonal antibodies, make sure they are of an isotype recognized by Protein A-might need to use Protein G beads instead (e.g. for anti-myc 9E10).

o Wash IP's
Try to get through these as quickly as possible.
1. Spin down beads @10K, 1min, RT.
2. Aspirate supernatant.
3. Wash with 1ml of the following buffers and mix by inverting 3 or 4 times. Only aspirate supernatant to 0.1ml marking so no beads are lost:
o FA lysis buffer (no supplements)
o FA wash buffer
o LiCl/Detergent
o TE
4. Wash a second time with TE:
o Wash with 500µl TE.
o Transfer beads and wash to second tube.
o Wash the old tube with 500µl TE and transfer to second tube.
o Elute

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