Chromosome conformation capture

3C Protocol

(Based on Louwers et al., 2009) modified by Junghyun Kim

1. Prepare plant tissues (around 1.5 g – 2 g)
2. Crosslinking (in 50 ml falcon tube)
   2% formaldehyde solution (20 ml per ea. sample)
   - 37% Formaldehyde 1,080 ul 
   - Add 1mM EDTA (pH8.0), 1mM PMSF, 1XPBS to 20ml
3. Dip the plant tissue to the crosslinking solution and mix well
   - Make sure that the plant tissue be under the solution (completely dipped into the solution) to increase crosslinking efficiency
4. Open the lid of the tube and vacuum for 25 min. (10 min -break- 10 min -break- 5 min)
5. Add 1 ml of 2 M glycine (final concentration to 0.125 M) and mix well for 5 min. vacuum and release.
6. Pour the solution and wash the crosslinked plant tissue 3 times with D.W.
7. Dry the plant tissue by placing them between paper towels.
8. Put the tissue to empty 50 ml falcon tube and freeze in liquid nitrogen. Keep it at -80 ^oC or grind it right away with mortar and pestle.
9. Add 10 ml of nuclei isolation buffer (ice-cold) to the #8 sample.
   Nuclei isolation buffer (10 ml)   *need 12 ml per one sample
   - 1 M HEPES (pH8.0) -> 200 ul (final 20 mM)
   - 2 M Sucrose -> 1.25 ml (final 250 mM)
   - 1 M MgCl2 -> 10 ul (final 1 mM)
   - 1 M KCl -> 50 ul (final 5 mM)
   - 100% Glycerol -> 4 ml (final 40%)
   - 20% TritonX-100 -> 175 ul (final 0.25%)
   - 100 mM PMSF -> 10 ul (final 0.1 mM)
   - 3.5 ul Beta-mercaptoethanol
   - Protease inhibitor cocktail
10. Gently mix and let them thaw on the ice.
11. Place a piece of Miracloth (4 layers) on top of a 50 ml falcon tube (making a filter) and add 1 ml of nuclei isolation buffer to the Miracloth (soaking before use). Pour the mixture through the filter.
12. Centrifuge for 15 min. at 3,000 g at 4 ^oC. Gently pour the supernatant and add 1 ml of nuclei isolation buffer and gently pipette up and down to resuspend the pellet.
13. Transfer the nuclei suspension to 1.5 ml EP tube and centrifuge for 5 min. at 1,900 g at 4 ^oC. And pipette off the supernatant.
     *need 1.4 ml 1.2X buffer per one sample
14. Resuspend the nuclei in 400 ul of 1.2 X restriction enzyme buffer. Centrifuge for 5 min at 1,900 g at 4^oC. Remove the supernatant and resuspend the pellet in 1 ml 1.2 X restriction enzyme buffer.
15. Add 30 ul of 10% SDS (final 0.3% SDS). Shake at 900 rpm for 40 min. at 65 ^oC and followed by 20 min. at 37 ^oC.
16. Add 100 ul of 20% Triton X-100 (final 2%). Shake at 900 rpm for 60 min. at 37 ^oC.
17. Add 400 U (8ul of 50,000U/ml) of the selected restriction enzyme (ex. DpnII) and incubate overnight at 37 ^oC while shaking at 900 rpm. (after overnight incubation, aliquot 10 ul for gel analysis)

==============2nd day==========

18. To inactivate the restriction enzyme, add 80 ul of 10 % SDS (final 0.8 %).

19. Incubate for 20-25 min. at 65 ^oC while shaking at 900 rpm.

20. Transfer the sample to a 50 ml falcon tube.

21. Add 3 ml of 1X ligation buffer (300 ul of 10 X Ligation buffer + 2.7 ml of D.W)

22. Add 200 ul of 20 % Triton X-100(final 1 %). Incubate for 1 hour at 37 ^oC.

23. Add 1 ul of T4 ligase highly concentrated (2,000,000U/ml) and incubate for 5 hours at 16 ^oC and followed by 2 hour at RT (or RT for 4~5 hrours) (Take 20 ul for gel analysis)

24. Add 15 ul of proteinase K (NEB) (total 300 ug).

25. Incubate the sample at 65 ^oC overnight to reverse the crosslinks.

==============3rd day============

DNA purification

26. Add 10 ul of RNase A (5 ug/ml) and incubate for 10 min. at 37 ^oC (*not necessary)

27. Add 10 ml of phenol-chloroform-isoamyl alcohol solution (pH 8.0 - Equilibrated) and mix well by rigorously shaking the tube by hand.

28. Centrifuge for 10 min. at 3,000 g at room temp.

29. Transfer the aqueous phase to a new falcon tube (3~3.5 ml).

30. Add 300 ul of 3M NaOAC (pH 5.2), 10 ul of glycogen and 7 ml of 96%(v/v) EtOH and mix well by inverting the tube several times.

31. Store at -80 ^oC for overnight. (keep at -80 ^oC for 1~ 2 days)

=============5th day============

32. Centrifuge the frozen tube for 60 min. at 3,000 g at 4 ^oC.

33. Discard the supernatant and wash the pellet with 10 ml of cold 70% EtOH.

34. Centrifuge for 15 min. at 3,000 g at 4 ^oC.

35. Pipette off as much liquid as possible and air dry the pellet for 3-5min

36. Add 150 ul of 10 mM Tris (pH 7.5) to the pellet.

37. To dissolve the DNA, incubate the pellet at room temperature for several hours followed by overnight incubation at 4 ^oC. -> save 5 ul for gel analysis qPCR and efficiency confirm by loading on the gel.

38. Use 1ul of DNA per qPCR.

#Plasmid 700~800 ng (Proceed together with 3C samples from enzyme cut step)

1. Digestion : DpnI cut -> 37 ^oC overnight incubation
    Final 50 ul – DpnI 1 ul       
                        Cutsmart buffer 5 ul
                        D.W. to final 50 ul

2. Inactivation: 20 min. at 80 ^oC

3. Ligation: Add 6 ul 10X T4 ligase buffer
                   Add 2 ul ligase (800 U), add 2 ul D.W. (Final 60 ul)

                  16 ^oC 5 hour, 45 min. RT (or RT for 4~5 hour)     

4. Dilute 10 X and use 1 ul for qPCR.

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