ChIP-Seq with Anti-HA
This protocol is adapted from Aparicio, O. et al (2005) Curr Protoc Mol Biol. Posted by Erbay Yigit on Oct-29-2008. Last modified on Apr-24-2009.
I. Cross-link protein-DNA complexes in vivo
- Grow yeast culture overnight. Next morning, inoculate 1 liter YPD, bring cell culture to ~1x10^6 cells per ml (OD600 0.1 Abs). Grow until the culture reaches mid log phase.
- Cross-link cells with 1% formaldehyde at room temperature for 20 minute (use 37% stock formaldehyde).
- Stop cross-linking with 0.125 M heat-sterilized Glycine, incubate another 5 minutes.
II. Harvest cells
- Collect cells by filtering cell culture through a 0.45 micron Nylon membrane filter or by centrifugation. Vortex the filter membrane in ice cold TBS to collect the cells.
1 liter yeast cell culture at OD600=1.0 Abs yield 1 g pellet.
- Centrifuge cells 5 min at 2,500 x g, 4°C. Discard supernatant into a chemical waste container and resuspend pellet in 25 ml ice-cold TBS. Repeat once.
Important: If you want to divide the cell pellet into aliquots, do so before washing it with Anti-HA Lysis buffer (next step). Otherwise, it is difficult to divide the pellet in Anti-HA Lysis buffer since it foams due to detergent.
- Resuspend cells in 10 ml ice-cold Anti-HA Lysis buffer.
- Pellet cells by centrifuging in a benchtop centrifuge 5 min at 3,000 x g, 4°C. Discard supernatant.
The cells can remain on ice for a few hours while other samples are collected. Alternatively, the cells may be frozen in liquid nitrogen for storage in -80°C.
III. Lyse cells and isolate chromatin
- Resuspend the cell pellet in 1 ml ice-cold Anti-HA lysis buffer/2 mM PMSF. Fill 1/2 or three-quarters
of a 2-ml flat-bottomed screw-cap microcentrifuge tube with 0.5-mm diameter
silica-zirconia or glass beads. Add cells, taking care to avoid introduction
of bubbles, and screw the cap on tightly. Make sure there are no leaks.
Silica-zirconia beads are more efficient
at breaking cells than glass beads and are recommended. To facilitate cell breakage
with the mini bead beater, it is important that the final suspension nearly fill the tube. Please follow manufacturer's suggestions for the best performance. For larger cultures, split the cells into multiple tubes. As a rule, I use 1 ml lysis buffer for 1 g of yeast cell pellet.
- Lyse cells 3 min with a mini bead beater (MBB) at maximum speed. Remove sample and
incubate 1 min in an ice-water bath. Repeat four times for a total breakage time of
15 min. If you are using bead beater (BB), 6 minutes lysing is enough. However, you must stop after every 1 min beating and keep the cells on ice at least 2-minutes.
This step assumes breakage with silica-zirconia beads. The cell breakage time with glass
beads may be longer.
IV. Isolate Lysate
- Cut a 5-ml syringe 1cm below the flared opening (i.e., where the plunger is inserted)
with a razor. Insert the smaller portion into a 15-ml disposable conical tube so that
the flared portion of the truncated syringe rests on top of the conical tube opening,
forming a microcentrifuge-tube holder.
- Invert the sample tube and punch a hole in the bottom with a 22-G or 25-G needle. Place the
sample tube into the syringe/conical tube and punch a hole in the top cover with the
same needle.
- Spin the assembly in a benchtop centrifuge 1 min at 1000 rpm, 4°C. Place the conical
tube on ice. Discard the 2-ml centrifuge tube containing the dry beads after confirming
the sample has been transferred to the 15-ml tube.
V. MNase digest DNA
- Transfer the sample to a standard 1.5-ml microcentrifuge tube. Microcentrifuge
20 min at maximum speed (22,000 x g), 4°C. Discard the supernatant and resuspend the pellet with ice-cold ***MNase reaction buffer***. Add enough buffer that would give 0.5 g cell pellet/ml concentration. If you make it more concentrated, MNase digestion does not look very good. I believe this is because chromatin is not very well dissolved at higher concentrations.
The pellet contains the cross-linked chromatin, cell debris, and unbroken cells. The purpose
of this centrifugation step is to remove soluble protein, most of which is not crosslinked
to DNA, as it might contribute to nonspecific background in the subsequent immunoprecipitations
step.
- Digest cross-linked chromatin by MNase. Stop digestion by adding EDTA at a concentration of 10 mM.
First, test digest a faction of cross-linked chromatin by MNase to find the best digestion conditions. Analyze it as described in Section VI, steps i, ii, and iii.
- Centrifuge the digested chromatin at ~13,000 x g for 15 minutes, 4°C.
Upon MNase digestion, the cross-linked chromatin is solubilized and purified away from the
pelleted material which contains cell debris and unbroken cells. The resulting chromatin
solution constitutes the input sample for the subsequent immunoprecipitation. Note that solubility of the mononucleosomal fraction depends on the salt concentration. In my hands, reducing the salt concentration three folds at this step by adding water increased the amount of mononucleosomes in the supernatant. The frozen
aliquots are stable for many months when stored at -80°C and are suitable for immunoprecipitations.
VI. Check MNase digested chromatin (Test digest)
- For each time point of digestion, do following:
Add equal volume of ChIP elution buffer to 250 microliter MNase digested chromatin aliquot.
Add Pronase from 20 mg/ml stock solution at a concentration of 0.8 mg/ml (20 microliter for 500 microliter sample volume).
Incubate 2 hr at 42°C, followed by 6 hr at 65°C.
- Extract with Phenol/Chloroform, and then with Chloroform (1x Vol).
No need for ethanol precipitation here! if you are careful enough not to pipet organic phase.
Add RNAase A (20 microg/ml) and incubate for half an hour or longer at 37°C. Extract with Phenol/Chloroform, and with Chloroform alone. Add 1/10 V of 3 M NaAcetate pH 5.2 and glycogen (optional) to precipitate.
Alternatively, you can concentrate your DNA with Millipore Centricon or Microcon devices instead of ethanol precipitation (I use YM-30 size membrane for mononucleosomal DNA size).
- Run an agarose gel to check MNase digested DNA.
VII. Immunoprecipitation
- Save the supernatant from step-15; bring Tris.HCl concentration to 50 mM, NaCl concentration to 150 mM, and NP40 concentration to 1.0% (to the levels in the Anti-HA lysis buffer).
- Add the Anti-HA resin to the protein extract.
- Incubate Anti-HA affinity matrix for 2 hours to overnight, +4 to +8°C
- Pellet Anti-HA matrix at full speed in a microcentrifuge for 5-10 sec or at 1,000 x g for 5 min.
- Wash twice with TBS-T (Tween-20 at 0.05%), and wash four times or more with TBS at 4°C.
VIII. Elution of HA-fusion protein with HA peptide (YPYDVPDYA)
- Prepare HA elution solution based on manufacturer's recommendation.
- Elute with HA peptide in TBS at 300 microgram/ml concentration for half an hour at room temperature (Sigma suggests elution at 100 microgram/ml concentration. You can try this lower concentration to save money on HA peptide).
- Pellet Anti-HA matrix at full speed in a microcentrifuge for 5-10 sec. Transfer the supernatants to
fresh test tubes using a Hamilton syringe or equivalent device. Be careful not to transfer
any resin.
- For immediate use, store the supernatants at
2-8°C. Store at -20°C for long term storage.
IX. Reverse cross-links and purify DNA
- Reverse cross-link and purify DNA as described at Section VI, steps i-iii.
Reagents and Solutions
Anti-HA Lysis Buffer for ChIP
| 50 mM |
Tris.HCl pH 7.5 |
| 150 mM |
NaCl |
| 1 mM |
EDTA |
| 1% |
NP-40 |
Store up to 1 year at room temperature. Freshly add 2 mM (f.c.) PMSF before use.
MNase reaction buffer
| 10 mM | Tris.Cl pH 7.5 |
| 10 mM | NaCl |
| 3 mM | MgCl2 |
| 1 mM | CaCl2 |
| 0.1 (v/v) | NP-40 |
Store buffer with above components up to 1 year at room temperature. Immediately before use, add 1 mM PMSF (fc) (from stock in ethanol).
ChIP elution buffer
| 50 mM | Tris.Cl pH 7.5 |
| 10 mM | EDTA |
| 1% | SDS |
Store up to 1 year at room temperature.