Chromatin Immunoprecipitation Protocol

Chromatin Immunoprecipitation Protocol (Yeast)
Harvesting Cells
1. Grow a 10 mL culture of yeast O/N at 37C in YPD
2. The next day, inoculate a 50-100 mL of media in a 250 mL flask to an OD
600
of ~0.3
a. Use selective media if maintaining a plasmid with a selective marker
3. Grow cells to an OD
600
of 0.800-1.000

Fixing Cells
4. Slowly add 37% Paraformaldehyde (in hood) to the cells to a final concentration of 1%
a. 1.35 mL for 50 mL sample
b. 2.70 mL for 100 mL sample
5. Shake at RT for 15 minutes at 100 rpm in shaker
6. Add glycine to a final concentration of 125 mM and shake sample again for 5 minutes at RT
7. Spin cells down in tabletop centrifuge at 4C for 5 minutes
8. Wash cells 2x with 30-40 mL of ice cold 1xTBS
9. Snap freeze cells
a. At this point you can freeze the cells O/N at -80C or keep going

Lysing the Cells
10. Resuspend the cells in 400 l of ChIP lysis buffer for every 50 mL of sample grown
11. Weigh out 0.5 grams of acid washed beads in a 1.5 mL microcentrifuge tube and place
resuspended sample in tube
12. Bead beat (using Smth lab mini-bead beater in cold room) 8x at 40 seconds ON and 1 minute
OFF
13. Poke a hole in the bottom of the tube with a hot 25 gauge needle and place the tube in another
1.5 mL tube
14. Spin the tubes for 5 minutes at 5000 rpm at 4C
a. You will have to leave the cap off of the rotor when spinning because the stacked tubes
will not fit under the cap
15. Make sure all of the cells and lysate has passed into the new tube. If not, wash the beads with
100 l of ChIP lysis buffer and spin into a new tube. You can recombine the wash with the
previous lysate.

Sonicating the cells
16. Resuspend the cells/debris and lysate and replenish protease inhibitors
17. Split resuspension into new 1.5 mL tubes containing NO MORE THAN 300 l of sample
a. For 50 mL, I usually get 2-3 tubes
b. For 100 mL, I usually get 4 tubes
18. Place samples in Bioruptor rotors
a. Each spot in the rotors should be filled (with blanks or samples) to give equal sonication
19. Sonicate using the Twin Bioruptor on HIGH 20x for 30 seconds ON/OFF
a. I change the water bath once in between the cycles so the bath doesnt get too warm
b. This means I do 10x water change 10x
20. Spin down the samples at 4C for 5 minutes at maximum speed
21. Transfer the supernatant to a new tube and repeat the spin for 15 minutes
22. Transfer the supernatant to a new tube
a. This is your lysate you will used for IP

Pre-clearing the Lysate
23. Determine your protein concentration using a BCA kit and BSA standards
24. Measure out ~1.0 mg of protein and add ChIP lysis buffer so final volume is 500 l
a. I have found that ~250 l of sample gives about 1.0 mg of protein
b. I usually just measure the OD
562
of the samples and use 250 l of the most
concentrated sample and adjust accordingly for the others
25. Add ~40 l of beads without antibody for preclearing
a. I use protein A beads for antibody/protein A IPs
b. I use Glutathione Sepharose for TAP/IgG IPs
26. Rotate at 4C for 2 hours

Immunoprecipitation
27. Spin down samples at 1000 g for 2 minutes at 4C
28. Transfer supernatant (DO NOT SUCK UP BEADS) to a new tube
29. Save 1/10
th
of the volume for input samples used in quantitative PCR
a. The volume should be ~50 l
b. Input can be kept at -20C until reversing crosslinks
30. Add antibody or IgG beads and rotate O/N at 4C
a. Add 5 g of primary antibody
b. Add ~40-50 l of IgG beads if ChIPping TAP tagged protein
31. The following day, add ~40-50 l of protein A beads to antibody/lysate mixture if you are using
a primary antibody
32. Rotate for 2 hours at 4C

Washing
33. Spin down the beads at 1000 g for 2 minutes at 4C
34. Decant supernatant
35. Wash beads at 4C in the following manner for 5 minutes each
a. 2x ChIP lysis buffer
b. 1x IP1
c. 1x IP2
d. 1x TE
e. 1x TE (at RT)

Reversing Crosslinking
36. Add 100 l of elution buffer and incubate at 65C for 15 minutes with brief vortexing every 5
minutes
37. Spin beads down at maximum speed and transfer elution to a new tube
38. Add ~70-80 l to beads and repeat elution
a. Final elution volume should be 200 l
39. DONT FORGET! Add 150 l of elution buffer to inputs from step 29
40. Leave samples O/N at 65C

DNA Purification
41. Add 800 l of binding buffer to samples and use Invitrogen PCR purification kit to isolate DNA
42. Measure DNA concentration of inputs
a. Mine are usually 40-100 ng/l

Quantitative PCR

I prepare 1:10 dilutions of my inputs and use 2 l of my samples and diluted inputs for each well. I
make five 5-fold dilution standards with undiluted input. You can use any input for the standards.
Below is an example of a plate with 2 primer sets and 2 samples (e.g. wild-type and mutant).Rows A
and B are standards. Rows C-F are sample measurements based off of the standards. I make
triplicate measurements. I left out half of the plate because it would not be used.

I add 2 l of my sample/standard to each well. I make a master mix with water, primers, and SYBR
green mix so my final reaction volume is 20 l with 100 nM primer concentration. I place a film on the
plate and vortex the plate in a PCR rack. I spin down the rack and plate at 3000 rpm for 4 minutes at
24C. The plate is then ready for PCR.

I use the relative enrichment method so I have a control region which does not contain my protein of
interest. To measure enrichment, I use the following equation:

CT = CT sample CT input
CT = CT
control
- CT
sample

Relative enrichment = (slope of standard curve)
CT

1 2 3 4 5 6
A
Primer
#1
Undiluted
input
1:5 dilution 1:25
dilution
1:125 dilution 1:625 dilution 1:3125 dilution
B
Primer
#2
Undiluted
input
1:5 dilution 1:25
dilution
1:125 dilution 1:625 dilution 1:3125 dilution
C
Primer
#1
WT Sample WT Sample WT Sample WT input 1:10
dil.
WT input 1:10
dil.
WT input 1:10
dil.
D
Primer
#1
Mutant
Sample
Mutant
Sample
Mutant
Sample
Mutant input
1:10 dil.
Mutant input
1:10 dil.
Mutant input
1:10 dil.
E
Primer
#2
WT Sample WT Sample WT Sample WT input 1:10
dil.
WT input 1:10
dil.
WT input 1:10
dil.
F
Primer
#2
Mutant
Sample
Mutant
Sample
Mutant
Sample
Mutant input
1:10 dil.
Mutant input
1:10 dil.
Mutant input
1:10 dil.
G
H
I

Solutions

All solutions are filtered sterilized. The lysis buffer, IP1, and IP2 are made fresh
for each ChIP.

Glycine - 2.5 M, pH 8.0, filter sterilized

ChIP Lysis Buffer - 50 mM HepesKOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton
X-100, 0.1% sodium deoxycholate, 1 mM PMSF, 1 g/ml leupeptin, 1g/ml pepstatin A

IP1 ChIP lysis buffer with 500 mM NaCl

IP2 10 mM TrisHCl, pH 8.0, 250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate,
1 mM EDTA

ChIP Elution Buffer 1.0% SDS, 10 mM Tris, 1 mM EDTA, pH 8.0

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