Fast ChIP[DNA | IP]

From "Protocol for the fast chromatin immunoprecipitation (ChIP) method," Nelson et al., Nature Protocols Vol.1 No.1 2006. Read the paper thoroughly before doing this ChIP protocol for the first time. Keep a copy of this paper handy, as it has useful details for troubleshooting etc.

2.5 x 10⁷ cells should be enough to yield 2 ml of sheared chromatin. If using 150 ul/IP (recommended), then this is enough for ~13 IPs. Then, each IP should yield enough DNA for 30+ 25-ul PCRs, or 80 10-ul PCRs.

Crosslinking and harvesting cells

1. Crosslink and harvest cells.

-Per IP, need 2 x 10⁶ cells.

-Crosslink with 1.42% formaldehyde (per ml medium, add 40 uL of 37% formaldehyde). (for 5 to 15 minutes).

2. Quench formaldehyde with glycine.

Add 1 M glycine (141 ul per ml of media) and shake gently for 5 minutes at room temperature.

3. Collect and wash cells.

-Wash twice with cold PBS, with protease inhibitors. (per ml PBS, add 5 ul of 0.1 mM PMSF and 10 ul of 1 ug/ul leupeptin). Use 15 ml conical tubes, and pellet by centrifugation at 2000g for 5 minutes at 4°.

***OK to stop here*** and freeze at -80° for months.

Lysis Must be done at 4° or on ice.

4. Lyse cells in IP buffer.

- Resuspend cells in IP buffer (with inhibitors), using 1 ml per ~ 6 x 10⁶ cells.

- Pipette up and down several times.

5. Isolate nuclei.

-Spin at 12,000g for 1 minute, 4°.

6. Wash pellet with IP buffer.

-Wash nuclear pellet with 1ml IP buffer containing inhibitors, and centrifuge as above.

Sonication

7. Resuspend pellet and sonicate.

-Resuspend pellet in 1 ml IP buffer (with inhibitors) per 10 million (1 x 10⁷) cells.

-Separate into 1-ml aliquots and sonicate on ice.

-Sonicate pellet (need to try different conditions for different cell types, with the goal of shearing to 0.5-1 kb chromatin fragments.

8. Clear lysate.

-Spin lysate at 12,000g 10 minutes 4°, and retain supernatant.

9. Set aside input DNA.

~0.2 million cells, or 10% of what will be used in IPs. This will be the ‘total DNA' sample (continued on step 19).

*** OK to stop here; freeze chromatin at -80° for months***

Immunoprecipitation

10. Set up immunoprecipitations.

-Aliquot sheard DNA into microfuge tubes. Each IP should contain the equivalent of 2 million cells (2 X 10⁶).

-Add antibody to IPs, and the same amoung of non-specific IgG to ‘mock IP' samples.

-(the amount will be different with each antibody).

-Incubate at 4°C O/N (or in ultrasonic waterbath for 15 minutes, if available)

***Critical: if using antibodies that do not bind to Protein-A sepharose (mouse and rat), set up a tube of Protein-A sepharose beads with Anti-rat IgG to be used in step 13***

11. Clear chromatin.

-Centrifuge to clear particulate matter from chromatin, at 12,000g for 10 minutes at 4°.

12. Wash the protein-A agarose beads.

-During steps 10 and 11, wash protein-A beads (20 ul per sample) 3 times with IP buffer to remove ethanol. Washes consist of resuspending beads with 1 ml of IP buffer, centrifuging (1000- 2000g) for a few seconds at room temperature, and aspiratingteh supernatant.

13. Aliquot beads into new tubes.

Dilute beads 1:1 with IP buffer and aliquot 40 ul slurry into clean tubes. ** Make sure to cut off tips, and to keep beads suspended while aliquoting to ensure an equal distribution of beads.**

14. Transfer cleared chromatin to tubes with beads.

Transfer the top 90% of the cleared chromatin to tubes containing beads. It is VERY important not to transfer any of the particulate matter from step 11, in order to minimize background.

15. Rotate at 4°C for 45 minutes, to allow antibody/chromatin complexes to bind to the beads.

***During this time, precipitate the total/input DNA with 2.5X-3X volumes of ethanol, then wash with 70% ethanol. During step 17, Chelex slurry will be added to the pellet.***

16. Wash beads with IP buffer.

Centrifuge the slurry at 1000-2000g for a few seconds (4°-25°C) and remove supernatant. Wash beads 5-6 times with 1 ml cold IP buffer, without inhibitors. A wash consists of resuspending the beads, centrifuging, and aspirating the supernatant.

DNA isolation (room temperature)

17. Add Chelex slurry.

Add 100 ul of 10% Chelex 100 slurry directly to the washed beads. (Cut tips, and make sure Chelex remains in suspension while pipetting... it settles quickly). (see step 19; prep input DNA and get ready to boil at the same time).

Also add 100 ul Chelex slurry to the input DNA pellets, and treat as for IP samples.

18. Vortex briefly.

19. Boil for 10 minutes.

20. Proteinase K treatment

Allow samples to cool. Add 2 ul of 10 ug/ul ProtK to each sample. Vortex, incubate at 55° for 30 minutes. Boil again, 10 minutes.

21. Remove condensate.

Centrifuge tubes at 12000g for 1 minute at 4°. Transfer 80 ul of supernatant to new tube; this is where your DNA is (be careful to avoid pellet).

22. Retrieve the rest of the DNA

Add 120 ul water to beads, vortex for 10 seconds, spin again, and transfer 120 ul to the tubes from step 21. You now have 200 ul of ChIP'd DNA for analysis by real-time PCR.

Realtime PCR analysis

23. Initially:

For 25 ul reactions:

6.25 ul of template (up to 25% of reaction can be template)

0.75 ul Forward oligo (10 uM)

0.75 ul Reverse oligo (10 uM)

12.5 ul SyberGreen mix

4.75 ul water

--------------------------------------

25 ul