QUANTITATIVE CHROMATIN IMMUNOPRECIPITATION
(ChIP) PROTOCOL FOR YEAST
Patrick M. Schaeffer, Strubin Lab
Last update by Rachel Maria Imoberdorf February 2004
This protocol has several modifications to the protocol described in StrahlBolsinger et al. (1997) and Kuras et al. (1999).
Strahl-Bolsinger, S., Hecht, A., Luo, K., and Grunstein, M. (1997). SIR2 and SIR4
interactions differ in core and extended telomeric heterochromatin in yeast. Genes
Dev. 11, 83-93.
Kuras, L., and Struhl, K. (1999). Binding of TBP to promoters in vivo is stimulated by
activators and requires Pol II holoenzyme. Nature 399, 609-13.
ChIP Buffers
FA-lysis buffer
50 mM HEPES-KOH, pH 7.5
140 mM NaCl
1 mM EDTA
1% Triton X-100
0.1% sodium deoxycholate (w/v)
FA500-lysis buffer
50 mM HEPES-KOH, pH 7.5
500 mM NaCl
1 mM EDTA
1% Triton X-100
0.1% sodium deoxycholate (w/v)
Buffer III
10 mM Tris-HCL, pH 8.0
1 mM EDTA
250 mM LiCl
1% NP-40
1% sodium deoxycholate (w/v)
Elution buffer B
50 mM Tris-HCL, pH 7.5
1% SDS
10 mM EDTA
Cross-linking
1/ Grow a 50 ml yeast culture at 30°C to a final density of 1 to 2 x 10 7 cells/ml.
2/ Add 1.65 ml formaldehyde (37% aqueous) directly to the culture to a final
concentration of 1.2%. Mix by swirling.
3/ Incubate for 10 min at 30°C under continuous gentle agitation in a rotary shaker
(The cross-linking time should be determined empirically for each protein and
should be as short as possible).
4/ Prepare a 50 ml conical tube containing 0.47 g glycine (330 mM final).
5/ Quench the formaldehyde by transferring the yeast culture in this tube. Mix well
and incubate for 5 min at RT. Invert the tube from time to time.
6/ Place the cell culture on ice for at least 5 min (cells can be left on ice for several
hours).
7/ Pellet cells in a clinical centrifuge at 2000 rpm for 5 min at 4°C, decant
supernatant.
8/ Wash the pellet twice with 25ml ice-cold Tris-buffered saline (TBS). Decant
supernatant.
9/ Resuspend cells in 1 ml FA-lysis buffer, transfer into a 1.5-ml Eppendorf tube,
pellet cells at 3000 rpm for 2 min at 4°C. Remove all supernatant.
 At this stage cells can be frozen in liquid nitrogen and stored at –70°C.
Cell Breakage
1/ Resuspend cells in 500 l ice-cold FA-lysis buffer and add PMSF to a final
concentration of 1 mM.
2/ Add an equal volume of acid-washed glass beads. Break the cells by vortexing
for 40 min at 4°C.
3/ Pierce tube bottom with a 0.45-mm needle, place into a new tube and recover
the extract by a short spin.
4/ Resuspend the pellet (which contains the chromatin and cell debris) by flicking
the tube a few times. Add SDS to a final concentration of 0.5%.
Chromatin Shearing
1/ Sonicate 6 times with 10 pulses using a Sonifier Cell Disruptor B-30 (settings:
duty 90, pulsed mode, output limit 2500). Cool on ice for at least 1 min between
each series of 10 pulses. Avoid foaming as this will reduce the efficiency of DNA
fragmentation.
2/ Pellet cell debris at 14000 rpm for 15 min, 4°C, and transfer the supernatant to a
new 1.5-ml Eppendorf tube.
3/ Sonicate again 3 times with 10 pulses as before.
4/ Centrifuge for 5 min. Transfer the supernatant to a new 1.5-ml Eppendorf tube.
Set aside 20 l of the extract to a new 1.5-ml tube. This will be referred as the
Input sample and can be stored at –20°C until use.
 Extracts may be frozen in liquid nitrogen at this point and stored at –70°C.
Immunoprecipitation
1/ Transfer 100 l of extract (corresponding to about 1 to 2 x 108 yeast cells) to a
new 1.5-ml tube. Add 800 l FA-lysis buffer, 30 l protein A-Sepharose beads
(pre-incubated for 30 min at RT with rocking in 500 l FA-lysis buffer containing 1
g of sonicated and denatured herring sperm DNA and 10 g of BSA, and then
washed twice with 1 ml FA-lysis buffer), and appropriate amounts of the relevant
or control antibodies (Ab).
2/ Incubate O/N at 4°C with gentle mixing on a head-over-tail rotator.
3/ Pellet the beads by centrifugation in a microfuge at 3000 rpm for 2 min. Remove
supernatant.
4/ Wash the beads as follows: 1x with 1 ml FA-lysis buffer
1x with 1 ml FA500-lysis buffer
1x with 1 ml Buffer III
2x with 1 ml Tris-EDTA pH 8.0
After the last wash remove as much liquid as possible.
5/ Elute the precipitate from the protein A-Sepharose beads by adding 100 l of
Elution buffer B. Vortex mildly. Incubate 10 min at 60°C.
6/ Pellet the beads by centrifugation in a microfuge at 3000 rpm for 2 min at RT.
Transfer the eluate to a new tube.
7/ Repeat steps 5 and 6. Pool the eluates.
Cross-link Reversal
1/ Add to the Input (from step 4 in Chromatin Shearing) and Eluate samples 200 l
of Tris-EDTA pH 8.0 and 3 l of proteinase K at 20 mg/ml. Incubate for 4-5 h (up
to O/N) at 65°C.
2/ Extract samples twice with phenol-chloroform and once with chloroform. Add
1/10 volume 3 M NaOAc (pH 5.2), 3.5 g of glycogen as carrier, and 2 volumes
of ethanol. Let precipitate for at least 1 h at –20°C.
3/ Spin down the ethanol precipitate at 14000 rpm for 10 min at 4°C. Remove the
supernatant (be very careful, the pellet does not stick very well to the side of the
tube).
4/ Wash with ice-cold 70% ethanol.
5/ Dissolve the Input DNA in 100 l and the immunoprecipitated DNA in 150 l of
water. Store at –70°C.
Quantification by real-time PCR
We use the Eurogentec qPCR core kit for SYBR Green I and the ABI PRISM 7700
Sequence Detection System. If you use another kit and/or machine you might have
to adjust the conditions.
Quantification of the immunoprecipitated DNA is based on a standard curve derived
from the serial dilutions of the Input DNA. For each primer pair, it is advisable to first
generate a standard curve with four 10-fold serial dilutions to verify linearity of the
PCR reaction. Subsequently, we routinely use only two dilutions (1/100 and
1/10,000).
Controls should include no-Ab control, non-specific Ab and, whenever possible,
untagged strain + specific Ab.
All PCR reactions are done in duplicate.
1/ Mix each primer pair and dilute to 0.6 M per primer in water.
2/ Serially dilute the Input DNA 1/100 and 1/10,000 into water just prior to use.
3/ PCR Reaction mix
SYBR Green I master mix (2x)
Primers (mix of 0.6 M each)
DNA sample
13 l
6 l
6 l
____
25 l
PCR Parameters
1 cycle
--
95°C for 10 min
40 cycles --
95°C for 15 sec
55°C for 30 sec
60°C for 60 sec
1 cycle
Hold at 4°C
--
When using the Eurogentec qPCR core kit for SYBR Green I it is critical to perform
the elongation step at 60°C (for the beauty of the curves!).
Subject the amplification product of each primer pair to melting point analysis and
subsequent gel electrophoresis to ensure specificity of amplification.
The PCR-amplification curve obtained with the immunoprecipitated DNA should be
comparable (same slope) to, and fall between the curves of the selected Input DNA
dilutions.