Chromatin Immunoprecipitation (ChIP) Using Drosophila Antennal and Brain Samples

1. Sufficiently suspend Dynabeads protein G by gently pipetting.

2. With scissors, cut off the tip of a 200-μL pipette tip, and then use it to transfer 40 μL of Dynabeads protein G into a 1.5-mL low-retention microcentrifuge tube.

• Using the cut tips for this transfer allows the beads to flow easily.

3. Wash the beads three times with cold ChIP buffer I as follows:

• i. Settle the beads with a DynaMag-2 magnet.

• ii. Remove the buffer with a pipet.

• iii. Take the tubes off the magnet.

• iv. Add 300 μL of cold ChIP buffer I to the beads.

• v. Resuspend the beads by gently pipetting.

  • During these steps, do not dry the beads. Avoid harsh pipetting or vigorous vortexing. After the final wash, do not settle the beads with the magnet until Step 7.

4. Centrifuge previously fixed and sheared Drosophila chromatin at 21,130g for 5 min.

• These samples were frozen in Step 20 of Protocol: Sample Preparation for Chromatin Immunoprecipitation (ChIP) from Drosophila Antennal and Brain Samples (Du and Volkan 2024a). Sheared chromatin from brains should be in a volume of ∼300 μL of ChIP lysis buffer, and sheared chromatin from antennae should be in a volume of ∼180 μL of ChIP lysis buffer.

5. Transfer the supernatant into a 2-mL low-retention microcentrifuge tube.

6. Add 1800 μL (to brain chromatin) or 1900 μL (to antenna chromatin) of ChIP buffer I to dilute the sample to 2.10 mL or 2.08 mL in total, so that the concentration of SDS from the ChIP lysis buffer is <0.1%.

7. Settle the beads from Step 3 with the magnet and remove the ChIP buffer I with a pipet.

8. Take the tubes off the magnet and resuspend the beads with 500 μL of the diluted chromatin sample from Step 6.

9. Transfer the beads back into the same tube containing the diluted chromatin.

10. Rock the mixture on a nutating mixer for 1 h at 4°C.

11. Settle beads with the magnet.

12. From the supernatant, remove 19.5 μL (2%) of precleared chromatin as the input, place it in a 1.5-mL low-retention tube, and store it at −20°C until the next day.

• Because the chromatin sample is split into two 975-μL samples in the next step, 19.5 μL represents 2% of the antibody or no-antibody samples.

13. Split the rest of the precleared chromatin into two 1.5-mL low-retention tubes.

• i. Transfer 975 μL of the chromatin into one tube labeled “antibody (ab).”

• ii. Transfer another 975 μL of the chromatin into another tube labeled “no-antibody (no-ab, or IgG).”

• iii. Add a suitable amount of antibody to the ab tube only and nothing to the no-ab tube (or add the same amount of IgG from the same species as the antibody being used; this is the mock control).

  • For brain or antennae samples, 5 μL of ChIP-grade antibodies for RNA polymerase II or H3K27ac is suitable. The amount of antibody can be adjusted based on the tissues and/or the quality of the antibodies.

14. Rock the tubes on a nutating mixer overnight for 12–16 h at 4°C.

15. With scissors, cut off the tip of a 200-μL pipette tip and then use it to transfer 30 μL of suspended Dynabeads protein G into each of two low-retention 1.5-mL tubes.

• One tube is for the ab sample and the other is for the no-ab sample.

• i. Wash each tube of beads three times with cold ChIP buffer I as in Step 3.

• ii. After the last wash, briefly centrifuge the tube (<700g) and settle the beads with the magnet.

• iii. Remove the supernatant and resuspend the beads with 500 μL of the chromatin/antibody samples from day 1.

• iv. Transfer the bead mixture into the same tube containing chromatin.

• v. Rock the mixture on a nutating mixer for 1 h at 4°C.

16. Settle the beads with the magnet, remove the supernatant, and take the tubes off the magnet.

17. Perform one short wash and then one long wash with cold wash buffer I for ChIP.

• i. For the short wash, quickly rinse beads once with 500 μL of cold wash buffer I for ChIP (low-salt). Settle the beads with the magnet immediately, remove the supernatant, and take the tubes off the magnet.

• ii. For the long wash, add another 500 μL of cold wash buffer I for ChIP (low-salt) and rock the samples on a nutator for 5 min.

• iii. Briefly centrifuge the tube (<700g), settle the beads with the magnet, remove the supernatant, and take the tubes off the magnet.

18. Perform both a short and long wash with cold wash buffer II for ChIP (high-salt) as in Step 17.

• When performing the long wash of wash buffer II, prepare wash buffer III.

19. Perform both a short and long wash with cold wash buffer III for ChIP as in Step 17.

• When doing the long wash of wash buffer III, prepare the ChIP elution buffer for Drosophila and warm up the elution buffer at 65°C in water bath.

20. Perform two short washes with 500 μL of cold 1× TE buffer.

• Fewer washes or a lower salt concentration can be used if the enrichment signal is too weak.

21. Briefly centrifuge the tube (<700g) and settle the beads with the magnet. Remove the TE buffer from the beads and take the tubes off the magnet.

22. Add 100 μL of prewarmed ChIP elution buffer for Drosophila to elute for 10 min at 65°C. Gently shake the tube to mix samples every 3 min.

23. Settle beads with the magnet, transfer the supernatant to a clean 1.5-mL tube, and take the tubes off the magnet.

24. Repeat Steps 22 and 23.

25. Combine the two eluates into the same tube. At the same time, take out the input tubes and thaw them at room temperature.

• Avoid creating air bubbles.

26. For the input tube, add 180 μL of ChIP elution buffer for Drosophila to bring the volume to 200 μL.

27. Add 8 μL of 5 m NaCl to the input and antibody and no-antibody elution tubes and mix well.

28. Seal the tubes with Parafilm. Incubate the tubes for 4–5 h or overnight at 65°C.

29. Add 24 μL of 10× TE buffer and 2 μL of RNase cocktail enzyme mix to each sample. Incubate for at least 30 min at 37°C.

30. Add 2 μL of 20 mg/mL proteinase K and incubate for at least 1 h at 65°C.

31. Follow the manual provided by the QIAquick PCR purification kit to purify samples.

32. Elute DNA with 50 μL of double-distilled H₂O.

• It is important to use column extraction at the end because residual contaminants may persist if only phenol-chloroform extraction is performed. These contaminants would inhibit quantitative PCR reactions.

• Proceed to Protocol: Performing Quantitative PCR after Chromatin Immunoprecipitation (ChIP) of Drosophila Antennal and Brain Samples (Du and Volkan 2024b).