[Arabidopsis] Re: chromatin immunoprecipitation

sasha preuss sasha.preuss at gmail.com
Tue Apr 25 10:59:55 EST 2006


I've got this protocl working fairly well, it's a compliation of
several of the protocols out there:

-Sasha Preuss

Plant Chromatin Immunoprecipitation
and ChIP/Chop
Sasha Preuss, Richard Lawrence; Pikaard lab, Washington University

This protocol is adapted from:
Gendrel, A. V., Lippman, Z., Yordan, C., Colot, V. & Martienssen, R. A.
(2002)         Science 297, 1871-1873
Nelson, J.D., Denisenko, O., Sova, P., and Bomsztyk, K. 2006. Fast
chromatin immunoprecipitation assay. Nucleic Acids Res 34(1): e2.


Reagents needed:

1. Grow 2 large petri plates (150 mm) of Arabidopsis seedlings on
sterile MS-agar, 4g/L phytogel, 1% sucrose, pH 5.2 8-12 days.

2. Harvest seedlings in one 50ml conical centrifuge tube for each
plate.

3. Rinse seedlings with 40 mls deionized (Milli-Q) water, gently, 2
times

4. Add 37 mls 1.0% formaldehyde

5. Stuff top of 50ml conical tube (containing the formaldehyde soaked
seedlings) with nylon mesh to keep the seedlings immersed during vacuum
infiltration (and aid later rinse steps) Also poke holes in the cap of
the conical tube and screw on the cap.

6. Vacuum infiltrate the seedlings for 10 minutes in a desiccator
attached to a vaccum pump.  You should pull in excess of 25 inches
mercury

7. Quench crosslinking by adding 2.5mls of 2M glycine ([Final] =
0.125M) and continuing vacuum infiltration for an additional 5 minutes.


8.  Rinse seedlings 2 times with Milli-Q (deionized) water, remove as
much water as possible.

9. Grind seedlings in liquid Nitrogen to a fine powder.

10. Add powder to 30mls of Extraction Buffer 1 in 50 ml conical tube,
vortex, place on ice.

11. Filter solution through 2 layers of Miracloth into an oak ridge
tube.

12. Centrifuge solution 20 minutes @ 4000rpm (1940 x g)  in a Beckman
JA-20 rotor.

13. Remove supernatant and resuspend pellet in 1 ml extraction buffer 2
(transfer to 1.5ml eppendorf tube)

14. Centrifuge top speed in microfuge (~14,000 x g) 10 minutes at 4
°C.

15. Remove supernatant and resuspend pellet in 300ul extraction buffer
3.

16. In a clean eppendorf tube, add 300ul extraction buffer 3.  Layer
resuspended pellet from step 15 on top of this 300 ul cushion.

17. Spin solution 1 hour @ top speed in microfuge at 4 °C.

18. Remove supernatant and resuspend chromatin pellet in 500 ul of
nuclei lysis buffer (keep on ice).  Pool all resuspended chromatin
samples of the same genotype or chemical treatment.

19. Sonicate chromatin solution on ice 4 times, 10 seconds each @ 40%
duty cycle, power setting 2 on a Branson sonifier (Kranz lab)-pause 1
min. between 10 sec. pulses.

20. Centrifuge the chromatin sample 10 minutes, top speed (14,000 x g)
at 4 °C in a microfuge. Transfer supernatant to new tube and spin 10
minutes, top speed at 4 C.  Remove supernatant (crude chromatin) to new
tube (chromatin can be frozen at -80C at this point).

21. Transfer (2) 100ul aliquots of chromatin to separate siliconized
microfuge tubes.  Add 900ul of ChIP dilution buffer to each tube
containing PMSF and protease inhibitors.  Using siliconized tubes
significantly reduces carry over of non-antibody bound chromatin.

22. Equilibrate Protein A agarose beads blocked with salmon sperm DNA
(Upstate Biotechnology) by rinsing 3x in 1 ml of ChIP dilution buffer.

23. Preclear each chromatin sample by adding 40ul of equilibrated
Protein A beads to each sample and rotating at 4 °C for 1 hour.

24. Spin samples of chromatin and beads at 0.8 RPM, 2 min. at 4 °C to
pellet beads.

25. Combine two 1000ul samples and split into 3 tubes (mock, K4, and
K9) of 600ul and one of 60ul (10 % input) using siliconized
micrcentrifuge tubes. Add desired antibodies to each tube and no
antibody to one tube (this is the mock control).  Typical antibodies
and amounts are:
H3 K9 dimethyl (Abcam) = 10 ul
H3 K4 trimethyl (Abcam) = 10 ul

26. Incubate chromatin plus antibodies on a rotating mixer wheel, 4
hours to overnight at 4 °C (4 hours works great - no reason to go
longer unless you need a break).

27. Collect immune complexes by adding 40ul of Protein A agarose beads
(equilibrated in ChIP dilution buffer) and rotating at 4 °C for 1
hour.

28. Pellet beads by centrifuging 0.8 RPM, 2 min. at 4 °C.

29. Wash with 1 ml of each of the following wash buffers 5 min. at 4
°C each:
Low Salt Wash Buffer
High Salt Wash Buffer
LiCl Wash Buffer
TE (2 times)

30. Remove residual TE

31. add 100ul 10% Chelex (10g/100ml water, from biorad), vortex (chelex
binding ions that are required for the action of nucleases, protecting
the DNA during boiling)

32. reverse protein DNA crosslinks by boiling 10 minutes, allow to cool
to room temp.

33. Add  1 ul  of 20mg/ml Proteinase K, incubate 30 min at 50 degrees C

34. boil 10 minutes

35. centrifuge, collect supernatent into new tube

36. add 100ul TE to pellet, vortex, centrifuge, collect and combine
supernatants

37.  For amplification of rDNA I typically use 2.5 ul DNA in a PCR with
26 cycles


Chip/Chop

Take DNA from chip reaction and digest with McrBC.  GTP is sensitive to
freeze thaw cycles.

10x NEB buffer 2      		2ul
100x BSA				0.2ul
100x GTP				0.2ul
DNA 				5 ul
McrBC				1 ul
Water				to 20 ul

Be sure to also do a -McrBC control

Incubate 3 hrs at 37 degrees C, kill reaction for 15 minutes at 65
Use 2.5ul in PCR for 28 cycles




plant ChIP solutions

Extraction Buffer 1
				for 100 ml:		for 250 ml:
0.4M sucrose 		20 ml 			2M 50ml
10mM Tris-HCl pH 8 	1 ml 1M 		2.5ml
5mM BME (2 (beta)-
mercaptoethanol)
				35 µl 14.3M		 87.5 ml
1mM PMSF 			1ml 0.1M		 2.5ml
+ Pis (protease inhibitors)
(1 mini-tablet for 10mls) H20 to volume

Extraction Buffer 2
				for 10 ml:
0.25M sucrose		 1.25 ml 2M
10mM Tris-HCl pH 8 	 100 µl 1M
10mM MgCl2 		 100 µl 1M
1% Triton X-100 		 100ul 100%
5mM BME			  3.5 µl 14.3M
1mM PMSF 			 100ul .1M
+ PIs
H20 to volume

Extraction Buffer 3
				for 10 ml:
1.7M sucrose 8.5 ml 2M
10mM Tris-HCl pH 8 100 µl 1M
0.15% Triton X-100 15ul 100%
2mM MgCl2 20 µl 1M
5mM BME 3.5 µl 14.3M
1mM PMSF 100ul .1M
+ PIs
H20 to volume

Nuclei Lysis Buffer
				for 5 ml:
50mM Tris-HCl ph 8 	0.25 ml 1M
10mM EDTA 		100 µl 0.5M
1% SDS 			0.5 ml 10%
1mM PMSF			  50ul .1M
+ PIs H20 to volume

ChIP Dilution Buffer
				for 10 ml:
1.1% Triton X-100		110 µl 100%
1.2mM EDTA  		24 µl 0.5 M
16.7mM Tris-HCl pH 8 	167 µl 1M
167mM NaCl 		334 µl 5M
H20 to volume


Low Salt Wash Buffer

				for 50 ml:
150mM NaCl		1.5 ml 5M
0.2% SDS 			1ml 10%
0.5% Triton X-100 		.25 ml 100%
2mM EDTA 			200 µl 0.5M
20mM Tris-HCl pH 8 	1 ml 1M
H20 to volume

High Salt Wash Buffer
				for 50 ml:
500mM NaC 		5ml 5M
0.2% SDS 			1 ml 10%
0.5% Triton X-100 		.25 ml 100%
2mM EDTA 			200 µl 0.5M
20mM Tris-HCl pH 8 	1 ml 1M
H20 to volume

LiCl Wash Buffer:
				 for 50 ml:
0.25M LiCl 			3.125 ml 4M
0.5% NP-40 		0.25 ml 100%
0.5% deoxycholate
sodium salt			0.25 g
1mM EDTA 			100 µl 0.5M
10mM Tris-HCl pH 8 	0.5 ml 1M
H20 to volume

TE Buffer:
				for 50 ml:
10mM Tris-HCl pH 8	0.5 ml 1M
1mM EDTA 			100 µl 0.5M
H20 to volume

Protease Inhibitors

we use sigma plant protease inhibitor cocktail
at 1:100 dilution, you may also combine the
components yourself
				per 10ml:
Antipain 2.5ug/ml 		2.5ul of 1mg/ml
Bestatin .5ug/ml 		5ul of 1mg/ml
Leupeptin .5ug/ml 		1ul of 5mg/ml
Pepstatin A 4ug/ml 	40ul of 1mg/ml 
(in MetOH)




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