Chromatin Immunoprecipitation (ChIPs) Cloning Protocol (Farnham Lab)

Day 1

1. Add formaldehyde directly to tissue culture media to a final concentration of 1%. We generally use 1 x 107 cells per antibody per timepoint. For cloning, multiple IPs are performed and pooled at later steps therefore we generally start with 1 x 108 cells. Incubate adherent cells on a shaking platform and suspension cells on a stir plate for 10 minutes at room temperature.

2. Stop the crosslinking reaction by adding glycine to a final concentration of 0.125 M. Continue to rock or spin at room temp for 5 minutes.

3. For adherent cells, pour off media and rinse plates twice with cold 1X PBS. For suspension cells, centrifuge and wash cell pellet twice with cold 1X PBS (for suspension cells then proceed to step 6).

4. For adherent cells, add an appropriate volume (we use 5 mls per 500 cm2 dish) of 1X PBS or a 20% 1X Trypsin-EDTA solution (0.05% trypsin final) in 1X PBS. Incubate at 370 C for 10 minutes if using trypsin (we have found this step useful for cells which are difficult to swell. Thus, for cell types that are easily swelled, this step may not be necessary).

5. Following addition of trypsin or PBS, scrape adherent cells from dishes. If you have used trypsin, inactivate the trypsin by adding a small amount of serum. Centrifuge scraped adherent or suspension cells and wash pellet once with 1X PBS plus PMSF (10 ul per ml).

6 Resuspend cell pellet in cell lysis buffer plus the protease inhibitors PMSF (10 ul per ml), aprotinin (1 ul per ml) and leupeptin (1 ul per ml). The final volume of cell lysis buffer should be sufficient so that there are no clumps of cells. Incubate on ice for 10 minutes. Cells can also be dounced on ice with a B dounce several times to aid in nuclei release.

7. Microfuge at 5,000 rpm for 5 minutes at 40 C to pellet the nuclei.

8. Resuspend nuclei in nuclei lysis buffer plus the same protease inhibitors as the cell lysis buffer. Incubate on ice for 10 minutes.

9. Sonicate chromatin to an average length of about 2 kb while keeping samples on ice (the time and number of pulses will vary depending on sonicator, cell type and extent of crosslinking. To aid in sonication, it may also be helpful to add 0.1 grams of glass bead (212-300 microns; Sigma) prior to sonication). For cloning purposes, a size range of 1 kb to 3 kb is desirable. After sonication, microfuge samples at 14,000 rmp for 10 minutes at 40 C. At this point, chromatin can be snap frozen in liquid nitrogen and stored at -700 C.

10. Carefully remove the supernatant and transfer to a new tube. Preclear chromatin by adding blocked Staph A cells (see preparation of staph A cells at end of protocol). Use 50 uls of preblocked Staph A cells.

11. Incubate on a rotating platform at 40 C for 15 minutes. Microfuge at 14,000 rpm for 4 minutes.

12. Transfer supernatant to a new tube and divide equally among your samples. If starting with 1 x 108 cells, then divide into 9 samples; 8 to be precipitated with the antibody used for cloning and 1 for a no antibody or preimmune serum control. We also include a "mock" sample which contains IP dilution buffer instead of chromatin (no antibody and mock are critical to control for nonspecific interactions and DNA contamination of IP and wash solutions). Adjust the final volume of each sample with IP dilution buffer plus protease inhibitors (add 2 times volume of IP dilution buffer). Sample volumes should be between 200 and 600 uls. Add 1 ug of antibody to each Ab sample (this may vary for individual antibodies).

13. Incubate on a rotating platform at 40 C overnight (or for at least 3 hours).

Day 2

14. If you are not using rabbit polyclonal antibodies (ie. monoclonal antibodies, ect), add 1 ug of an appropriate secondary antibody (rabbit) and incubate for an additional 1 hour.

15. Add 10 uls of blocked Staph A cells to each sample. Incubate on the rotating platform at 4 0C for 15 minutes, no longer.

16. Microfuge samples at 14,000 rpm for 4 minutes. Save the supernatant from the "no antibody" sample as "total input chromatin".

17. Wash pellets twice with 1.2 mls of 1X dialysis buffer (**if you are using a monoclonal antibody, omit the sarkosyl**) and four times with 1.2 mls of IP wash buffer (**pH 8.0 for monoclonal antibodies**). For each wash, dissolve the pellet in 600 uls of buffer. Add an additional 600 ml of buffer then incubate samples on a rotating platform for 3 minutes. Next, microfuge samples at 14,000 rmp for 4 minutes. Try to remove as much buffer as possible after each wash without aspirating the Staph A cells.

18. After the last wash, microfuge and remove the last traces of buffer. Elute antibody/protein/DNA complexes by adding 30 uls of IP elution buffer. Shake on vortexer for at least 30 minutes at setting "vortex 3". Microfuge at 14,000 rpm for 4 minutes. Transfer supernatants to new tubes. (*Note: for the no antibody and mock samples only, omit steps 18-24 and elute as described in steps 25-26).

19. Microfuge collected samples at 14,000 rpm for 4 minutes to remove any traces of Staph A cells. Transfer supernatants to new tubes. Save the no antibody, mock, and total samples at 40 C until crosslink reversal step.

20. Add 270 uls of IP dilution buffer (plus protease inhibitors) for each 30 uls of eluted samples. Combine samples into 600 uls per tube for a total of 4 samples (if starting with 8 IPs). Add 1 ug of the same antibody used in the first precipitation and incubate at 40 C overnight (or for at least 3 hours).

Day 3

21. If you are not using rabbit polyclonal antibodies (ie. monoclonal antibodies, ect), add 1 ug of an appropriate secondary antibody (rabbit) and incubate for an additional 1 hour.

22. Add 10 uls of blocked Staph A cells to each sample. Incubate on the rotating platform at 4 0C for 15 minutes, no longer.

23. Microfuge samples at 14,000 rpm for 4 minutes.

24. Wash pellets twice with 1.2 mls of 1X dialysis buffer (**if you are using a monoclonal antibody, omit the sarkosyl**) and four times with 1.2 mls of IP wash buffer (**pH 8.0 for monoclonal antibodies**). For each wash, dissolve the pellet in 600 uls of buffer. Add an additional 600 ml of buffer then incubate samples on a rotating platform for 3 minutes. Next, microfuge samples at 14,000 rpm for 4 minutes. Try to remove as much buffer as possible after each wash without aspirating the Staph A cells.

25. After the last wash, microfuge and remove the last traces of buffer. Elute antibody/protein/DNA complexes by adding 150 uls of IP elution buffer. Shake on vortexer for at least 15 minutes at setting "vortex 3". Microfuge at 14,000 rpm for 4 minutes. Transfer supernatants to new tubes. Repeat and combine both elutions in the same tube.

26. After the second elution, microfuge samples at 14,000 rpm for 4 minutes to remove any traces of Staph A cells. Transfer supernatants to new tubes. Add 1 ul of high concentration RNase A (10 mg per ml) and 12 ul of 5M NaCl to a final concentration of 0.3 M. (Remember to include no antibody, mock, and “total” sample at this point to reverse crosslinks. For the total sample, use 20% of starting volume and increase the volume of the sample to 600 ul with elution buffer. Add 24 ul of 5M NaCl and 2 ul RNase to the total sample). Incubate samples in the 670 C waterbath for 4-5 hours to reverse formaldehyde crosslinks. After 4-5 hours, add 2 and a half volumes of ethanol and precipitate at -200 C overnight.

Day 4

27. Microfuge samples at 14,000 rpm for 15-20 minutes at 40 C. Respin and remove residual ethanol. Allow pellets to air dry completely.

28. Dissolve each pellet in 100 uls of TE. Add 25 uls of 5X PK buffer and 1.5 uls of proteinase K to each sample. The "total" sample will be viscous and may have to be dissolved in a larger volume. Incubate in 450 C waterbath for 1-2 hours.

29. Add 175 uls of TE to each sample. Extract once with 300 uls of phenol/chloroform/ isoamyl alcohol and once with 300 ul chloroform/isoamyl alcohol. Total input samples may need to be extracted twice.

30. Add 30 uls of 5M NaCl, 5 ugs of glycogen, and 750 uls of ethanol to each sample. Precipitate in -200 C freezer overnight.

Day 5

31. Microfuge samples at 14,000 rpm for 20 minutes at 40 C. Allow pellets to air dry. Resuspend DNA in 30 uls water and combine the 4 IP samples into 1 tube. Save an aliqout (10-15 uls) to analyze known targets by PCR to confirm the efficiency of the IP. Dilute the "total" sample in 30 uls; for PCR dilute the total an additional 100 fold and use 2-3 uls for each PCR reaction.

32. To create blunt-ended DNA fragments for cloning purposes, a DNA polymerase reaction is necessary. Add 60 uls of the chromatin sample, 20 uls of 1 mM dNTPs, 20 uls of 10X T4 DNA polymerase buffer, 2 uls 100X BSA, 92 uls of water, and 6 uls of T4 DNA polymerase. Incubate at 370 C for 30 minutes. (Depending on the concentration of the DNA in the precipitated sample, the amount of sample added to the polymerase reaction may need to be varied).

33. Perform a phenol:chloroform extraction on the sample. Add 200 uls of phenol:chloroform:isoamyl alcohol (25:24:1) and vortex. Spin samples at 14,000 rpm for 5 minutes. Save the aqueous phase to a new tube.

34. Extract the samples with chloroform. Add 200 uls of chloroform:isoamyl alcohol (24:1) and vortex. Spin samples at 14,000 rpm for 5 minutes. Save the aqueous phase to a new tube.

35. Precipitate the DNA with 1:10 volume 3M NaOAc and 2.5 volumes ethanol. Incubate samples at –200 C for at least 2 hours or on dry ice for 10 minutes.

36. Spin samples at 14,000 rpm for 10-20 minutes. Discard supernatant and dry pellet. Resuspend pellet in 10 uls of water.

37. Ligate the samples into a vector previously digested with an enzyme creating blunt ends. (For this step, we have used the pUC19 vector digested with Hinc II. Following digestion, the vector was treated with alkaline phosphatase to help prevent the religation of the vector without insert). Ligation reactions were performed in 10 uls volume and a range of chromatin concentrations were used (2 uls, 1 uls, 0.5 uls, and 0.1 uls). Ligations were performed at 160 C overnight.

Day 6

38. Perform transformation reactions using entire ligation mixture and competent bacterial cells. We have used TOP10 competent cells (Invitrogen) and a heat shock method for transformation. Plate cells under appropriate selection and incubate overnight at 370 C.

Day 7

39. Pick colonies and grow for minipreps. (Cultures can be grown for a minimum of 6-8 hours or overnight). Isolate vector using a miniprep plasmid purification protocol (such as those provided by Qiagen). Digest the vector with appropriate restriction enzymes to release the cloned DNA insert. (Remember that the blunted site that was cloned into will be destroyed). Run the digest on a 1% agarose gel to determine the insert size. We have found that cloned fragments of 500 bp or greater have been more likely to contain real clones and not just nonspecific DNA such as repeat regions, therefore we only proceed to analyze cloned inserts greater than 500 bp.

Day 8-10

40. Sequence the cloned inserts using vector specific primers (it is best to sequence the larger inserts from both directions).

41. Design clone-specific primers to analyze in independent chromatin immunoprecipitation experiments to confirm that the cloned fragment is specific to the antibody treatment and not just nonspecifically associated with the reactions.

Solutions

Cell Lysis buffer

5 mM PIPES pH 8.0

85 mM KCL

0.5% NP40

protease inhibitors

Nuclei Lysis buffer

50 mM Tris-Cl pH 8.1

10 mM EDTA

1% SDS

protease inhibitors

IP Dilution buffer

0.01% SDS

1.1% Triton X 100

1.2 mM EDTA

16.7 mM Tris-Cl pH 8.1

167 mM NaCl

1X Dialysis buffer

2 mM EDTA

50 mM Tris-Cl pH 8.0

0.2 % Sarkosyl (omit for monoclonal antibodies)

IP Wash buffer

100 mM Tris-Cl pH 9.0 (8.0 for monoclonal antibodies)

500 mM LiCl

1% NP40

1% deoxycholic acid

Elution buffer

50 mM NaHCO3

1% SDS

5X PK buffer

50 mM Tris-Cl pH 7.5

25 mM EDTA

1.25% SDS

Protease Inhibitors

100 mM PMSF in isopropanol, use at 1:100

10 mg per ml aprotinin in 0.01 M HEPES pH 8.0, use at 1:1,000

10 mg per ml leupeptin in water, use at 1:1,000

Staph A Cells

Resuspend 1 gram of lyophilized Staph A cells (Boehringer Mannheim) in 10 mls of 1X dialysis buffer. Centifuge at 10,000 rpm for 5 minutes at 40 C. Repeat. Resuspend in 3 mls of 1X PBS plus 3% SDS and 10% BME. Boil for 30 minutes. Centifuge at 10,000 rpm for 5 minutes. Wash in 1X dialysis buffer and cetrifuge at 10,000 rpm for 5 minutes. Repeat. Resuspend in 4 mls of 1X dialysis buffer. Divide into 100 ul aliquots, snap freeze and store in liquid nitrogen. Thaw 1 tube (100 uls) of cells for approximately every 108 cells that you begin with. Add 10 of herring sperm DNA (10 mg/ml) and 10 uls of BSA (10 mg/ml) to each tube of Staph A cells. Incubate on the rotating platform at 40 C for at least 3 hours, overnight is fine. Before using, microfuge for 3 minutes. Remove supernatant and wash pellet twice in 1X dialysis buffer. Resuspend cells in a volume of 1X dialysis equal to the original starting volume.

Note: in the protocol, the letter u is used to denote micro. This was done to prevent the loss of the symbol during downloading of the protocol in different programs.