Chromatin Immunoprecipitation Assay and PCR

Chromatin Immunoprecipitation Assay and PCR

(galactose inducible genes)

adapted by Wendy Reeves (Hahn Lab)from Marnie Gelbart (Tsukiyama lab)

Chromatin Preparation

Two days before prep,inoculate 5ml media (YP+ 2% Raffinose).30 ºC 24 hrs.

Day before prep,inoculate 300 ml media (YP+ 2% Raffinose)with overnight culture.

For YP-Raffinose,300μl wt will reach OD₆₀₀=0.4 in ~16 hours.

Day 1

For galactose induction,add 15 ml 40% galactose at OD=0.4.Grow one hour.

At OD=0.5,add 1/10 volume of newly made Fix solution.

Shake slowly at RT for 5-60 minutes.

- For Flag-Ste12,I crosslinked for 60 minutes

Add 18 ml 2.5 M Glycine per 100 ml culture

Shake at RT for 5 minutes

Pellet cells (GS3,5 min,6000g)

wash twice with 10 ml ice cold TBS in 15 ml screwcap tubes

- if necessary,cells can be frozen at this step.

Resuspend cells in 500μl Breaking Buffer⁺ 1 mM PMSF

transfer to flat bottom 2 ml microcentrifuge tube

add ~500μl glass beads

vortex in cold room,full speed,for 40 minutes

With 20 gauge needle,puncture bottom and then top of tube.

place tube immediately into 15 ml screwcap tube

spin 1000 rpm,2 min in tabletop centrifuge

Add 1 ml FA buffer⁺ protease inhibitors

transfer to 2 ml flat bottom tube

14k 1 min 4ºC

remove supernatant carefully with pipette

Wash pellet twice with 1 ml FA buffer+ PIs

(you should resuspend pellet by gentle pipetting)

Resuspend chromatin pellet in 300μl FA⁺ PI's

sonicate with microtip: 12x 10 sec at 1.5 setting with 2 minute breaks on ice between each round of sonication

Add 1.2 ml FA⁺ PIs and mix gently

spin 14 k,30 min,4 ºC

turn tubes and repeat 30 minute spin.(this gives you tight pellet)

Save 20μl to check shearing and freeze rest of supernatant (chromatin sample)by freezing in liq.N₂ and storing at -70.

Add 70μl H₂O and 90μl 2x Stop Buffer to 20μl of chromatin.

add 1μl (20 micrograms/microliter)glycogen

Incubate at 75 ºC (in hybridization oven)overnight.

Day 2

Add 20 micrograms proteinase K to chromatin

incubate at 55 ºC (hybridization oven)for at least three hours.

Phenol extract once

Phenol/chloroform extract once

EtOH precipitate.Wash once with 80% ethanol and dry briefly in speedvac.

Resuspend in 30μl TE⁺ 10 micrograms RNase A 37 ºC 1 hr.

Add 20μl TE and purify with invitrogen PCR cleanup kit.Elute in 50μl elution buffer.

Run 15μl on agarose gel to check shearing.Should be centered around 300-600 bp.

Immunoprecipitation

Prepare beads^- Day before immunoprecipitation

make 7 ml of 5 mg/ml IgG-free BSA in PBS fresh

Pipette 20μl magnetic Protein G beads per IP into siliconized eppie.

(make at least 80μl to ensure proper mixing during overnight incubation)

Wash with 3 times with 1 ml BSA solution^- rotate on nutator for 5 min at RT each time

Add 100μl/IP of BSA solution and resuspend well

add 1μl/IP of anti-Flag M2 antibody (~4 mg/μl)

Rotate overnight at 4 ºC (usually use Toshi lab's rotator)

Immunoprecipitation

Wash beads twice with 1 ml BSA solution as above

resuspend in 30μl/IP of BSA solution and keep on ice

Thaw chromatin quickly in RT water bath and aliquot amount needed

spin 14k 15 minutes 4ºC

- for a single IP,I usually take 250μl (200 for IP and 20μl for input DNA and,if desired,western analysis)

- refreeze rest of chromatin in liquid nitrogen

Filter chromatin:

prewet Millipore Ultrafree-MC 0.45 ºm filter unit with 50μl FA buffer

spin 14k 1 min 4ºC and discard wash

apply chromatin to filter and spin 14k 1 min 4ºC

save 20μl for input DNA and,if desired)20μl for western blotting

Add 200μl filtered chromatin to 30μl Dynabeads

rotate 90 minutes at room temperature

Wash beads (1 ml buffer,5 minutes on nutator,room temperature)

3 times with FA

2 times with FA-HS (high salt)

1 time with RIPA

Elute:

resuspend beads in 10μl RIPA containing 3x-Flag peptide (5 mg/ml) add 40μl RIPA 30 min.room temperature.1200 rpm in thermomixer repeat elution and combine.

if desired,save 10μl for western blot continue with DNA preparation or freeze in liquid nitrogen DNA preparation:

add equal volume (90μl)of 2x stop buffer to eluted DNA.

add 70μl H₂0 and 90μl 2x stop buffer to input DNA.

add 1μl glycogen (20μg/ºl)to all samples

incubate at 75 ºC overnight in hybridization oven.

add 20μg proteinase K (self-digested)

incubate at 55 ºC for at least three hours.

{alternatively,can incubate at 75 ºC for 6 hours and 55 ºC overnight}

Phenol extract once

Phenol/chloroform extract once

EtOH precipitate,wash once with 80% ethanol and dry briefly in speedvac

Resuspend in 30μl TE and 10μg RNase A incubate 37 ºC for 1 hour

Add 20μl TE and purify using invitrogen PCR cleanup kit elute in 50μl kit elution buffer.

Reagents:

Fix Solution (make fresh on day of crosslinking)30 ml

11 % formaldehyde 8.9 ml 37 % Formaldehyde

100 mM NaCl₀.6 ml 5 M NaCl

1 mM EDTA 0.12 ml 0.25 M EDTA

50 mM Hepes pH 7.6 1.5 ml 1 M Hepes pH 7.6

2.5 M Glycine

TBS 500 ml

20 mM Tris pH 7.6 10 ml 1 M Tris pH 7.6

150 mM NaCl₁5 ml 5 M NaCl

Breaking Buffer 2.5 ml

20 mM Tris pH 8.0 0.25 ml 1 M Tris pH 8.0

150 mM NaCl₁ ml 50 % glycerol

- add 1 mM PMSF fresh on day of chromatin prep.

FA Buffer 500 ml

50 mM Hepes pH 7.6 25 ml 1 M Hepes pH 7.6

150 mM NaCl₁5 ml 5 M NaCl

1 mM EDTA 2 ml 0.25 M EDTA

1 % TritonX-100 5 ml 100 % TritonX-100

0.1 % sodium deoxycholate 0.5 g sodium deoxycholate

FA-HS Buffer 500 ml

as above,except add 50 ml of 5 M NaCl instead of 15 ml.

RIPA Buffer 500 ml

10 mM Tris pH 8.0 5 ml 1 M Tris pH 8.0

250 mmliCl₂5 ml 1 mliCl

1 mM EDTA 2 ml 0.25 M EDTA

0.5 % NP-40 2.5 ml 100 % NP-40

0.5 % sodium deoxycholate 2.5 g sodium deoxycholate

2x Stop buffer 50 ml

20 mM Tris pH 8.0 1 ml 1 M Tris pH 8.0

100 mM NaCl₁ ml 5 M Nacl

20 mM EDTA 4 ml 0.25 M EDTA

1 % SDS 2.5 ml 20 % SDS

PCR:

For positive control,use a serial dilution of “input”.Exact dilution series will depend upon your IP signal strength.A reasonable starting series is 1:50,1:250,1:750.

For bound (IP)use undiluted DNA.

For primers: aim for PCR products around 200 - 350 bps and ann.temp.near 55 ºC

Reaction (20μl)

2μl 10x PCR buffer

1.6μl 25 mM MgCl₂

20 pmol primer 1

20 pmol primer 2

0.4μl 10mM dNTPs

0.4μl DMSO

0.4μl Platinum Taq (or similar heat activated polymerase)

2μl DNA sample

H₂O to 20℃

PCR protocol

95 ºC 2 min^- [95 ºC 20 sec^- 55 ºC 40 sec^- 72 ºC 30 sec]23-25 cycles^- 72 ºC 5 min.

Cold PCR: exactly as above and run reaction on 2 % agarose

Hot PCR (necessary for quantitation):

add 0.1μl [±-P32] CTP.(10 mCi/ml)to each reaction

after reaction,add 4μl 5x DNA loading buffer (same as for agarose gels)

run half of sample on 6% Acrylamide gel with 1x TBE running buffer

6% Acrylamide gel

26.25 ml H₂O

7 ml 30%/0.8% Acrylamide

1.75 ml 10x TBE

0.35 ml 10% APS

0.028 ml TEMED