Chromatin IP
Josh Friedman
12/02
1. Thaw frozen chromatin quickly at 37C
2. Spin in microfuge, max RPM x 5 min. Take supernatant, leave cellular debris.
You will use 2 µg chromatin DNA and 2 ug antibody per IP (determine empirically).
3. Pre-clear Chromatin: Add 125 µl 50% protein-A or protein-G agarose to each IP
(choice of protein-A or –G depends on origin of antibody). Dilute with ChIP
dilution buffer up to 1 ml and rotate 1 hour at 4C.
Eg: Chromatin is at 50 ng/ul
40 ul of chromatin (2ug)
125 ul Protein G agarose
835 ul of ChIP dilution buffer
4. Spin (all spins 15 sec pulse in microfuge) and use supernatant in IP (agarose is at
bottom, discard that part).
5. Add antibody: 1-4 µg affinity purified or monoclonal and rotate overnight at 4C
6. Make Blocked protein-A/G overnight with BSA and fish DNA in ChIP dilution
buffer. Final amounts:
50 ul Protein A/G agarose per IP
BSA to 1mg/ml
fish DNA to 0.1mg/ml
Eg: 6 samples
300 ul protein A/G agarose
100 ul of 10 mg/ml BSA
10 ul of 10mg/ml fish DNA (salmon sperm)
590 ul of ChIP dilution buffer
Rotate o/n at 4C. Do this on same day as setting up o/n IP.
Next day:
7. Before using blocked protein-A/G agarose, wash once with ChIP dilution buffer
by spinning down, discarding supernatant. Resuspend to 1ml with ChIP dilution
buffer.
8. Precipitate: add equivalent of 50 ul protein-A/G agarose to chromatin and rotate at
4C for 30-60 minutes.
Eg: 6 samples of blocked protein A/G agarose in 1 ml ChIP dilution buffer:
Use 1 ml / 6 = ~160 ul of Resuspended Protein A/G agarose. Mix well
between withdrawals.
9. Wash: 15 sec pulse to pellet agarose, remove supernatant, add 1 ml wash buffer,
rotate 5 min at RT
a. Wash buffers, in order: TSE I, TSE II, ChIP buffer III, TE
10. Elute: add 100 µl freshly-made ChIP elution buffer to final pellet and rotate 10
min at RT. Spin, keep supernatant. Repeat elution and combine eluates.
11. Un-crosslink: add 8 µl 5M NaCl per 200 µl eluate (final concentration 192 mM
NaCl) and incubate at 65C overnight. Cap tubes carefully or else you will get
evaporation.
Chromatin IP
Josh Friedman
12/02
12. Add 8 µl 1 M Tris-Hcl, pH 6.5, 4 µl 0.5 M EDTA, and 1 µl 10 mg/ml proteinase
K. Incubate for 1 hour at 45C. Qiaquick column purification (Qiagen), elute in
30 ul EB.
13. Do Q-PCR and quantify enrichment relative to non-specific genomic sequence.
For example, use primers to the 28S gene as a non-specific sequence, and
calculate enrichment from CT’s as follows: 2^[(28SChIP - YourGeneChIP) - (28SInput
- YourGeneInput)].
1. ChIP dilution buffer: 0.01%SDS, 1.1% Triton-X 100, 167 mM NaCl, 16.7 mM
Tris-HCl, pH 8.1
a. To make 50 ml:
i. 50 µl 10% SDS
ii. 5.5 ml 10% Triton-X 100
iii. 835 µl 1M Tris-HCl, pH 8.1
iv. 1.67 ml 5M NaCl
2. TSE I: 0.1% SDS, 1% Triton X-100, 2mM EDTA, 20 mM Tris-HCl, pH 8.1, 150
mM NaCl
a. To make 50 ml:
i. 500 µl 10% SDS
ii. 5 ml 10% Triton X-100
iii. 200 µl 0.5M EDTA
iv. 1 ml 1M Tris-HC, pH 8.1
v. 1.5 ml 5M NaCl
3. TSE II: 0.1% SDS, 1% Triton X-100, 2mM EDTA, 20 mM Tris-HCl, pH 8.1,
500 mM NaCl
a. To make 50 ml:
i. 500 µl 10% SDS
ii. 5 ml 10% Triton X-100
iii. 200 µl 0.5M EDTA
iv. 1 ml 1M Tris-HCL, pH 8.1
v. 5 ml 5M NaCl
4. ChIP Buffer III: 0.25M LiCl, 1% NP-40, 1% deoxycholate, 1mM EDTA, 10
mM Tris-HCl, pH 8.1
a. To make 50 ml:
i. 2.5 ml 5M LiCl
ii. 5 ml 10% NP-40
iii. 500 mg deoxycholic acid
iv. 100 µl 0.5M EDTA
v. 500 µl 1M Tris-HCl, pH 8.1
5. Elution buffer: 1% SDS, 0.1 M NHCO3
a. To make 5 ml:
i. Add 420 mg NaHCO3 to 45 ml water
ii. Take 4.5 ml of this and add 0.5 ml 10% SDS