Native chromatin immunoprecipitation (ChIP)

© Christoph Grunau, 2004,

This protocol is based on the protocol of David Umlauf, with contributions of Jerome Buard.

Before everything begins: prepare solutions...

• 1 M KCl, autoclave
• 5 M NaCl, autoclave
• 1 M MgCl, autoclave
• 1 M Tris/Cl pH 7.4 - 7.6, autoclave
• 0.5 M EDTA, autoclave
• 1 M CaCl2, 10 ml, sterile filter or autoclave
• 100 mM DTT, 1ml (store at -20°C)
• 1.8 g/l Aprotinin (Sigma A1153 5mg) (store at 4°C)
• 2.5 M Sodium butyrate (Sigma B5887 1g) (store at 4°C)
• 25 mM PMSF in isopropanol, 10 ml (store at -20°C)
• 15 U/µl Micrococccal nuclease (MNase) (USB 70196Y) in sterile 50% glycerol, aliquot to ~10µl and store at -20°C
• Protein A - sepharose CL-4B (Sigma P3391 250mg) (store at 4°C)
• agarose gel loading buffer
• 20% SDS
• 20 g/l glycogen solution (store at -20°C)

day 1...

• prepare a 2% 1x SB gel with 20 µl slots
• preheat a water bath to exactly 37°C
• prepare the following solutions with autoclaved distilled water

buffer 2
10 ml	buffer 1 (0.3 M sucrose)
40 µl (for tissue 80µl)	NP40 (cut pipette tip)
put on 37°C to fully dissolve NP40 and put on ice

buffer 3 (1.2 M sucrose) for 3 cell samples
20.55 g	sucrose
25 ml	2x base buffer
100 µl	Aprotinin
100 µl	sodium butyrate
200 µl	PMSF
250 µl	DTT
to 50 ml	water

MNase digestion buffer
1.1 g	sucrose
0.5 ml	Tris/Cl
80 µl	PMSF
40 µl	MgCl2
20 µl	sodium butyrate
10 µl	CaCl2 (essential for the enzyme)
to 10 ml	water

• put all buffer solutions on ice (except MNase buffer)
  
• cell lysis
  • prepare 1x10⁷ cells in PBS (cells can be stored in liquid nitrogen in 1 ml PBS) in a 2 ml Eppendorf tube
  • centrifuge 2500 rpm 10 min 4°C
  • remove supernatant
  • resuspend pellet in1 ml buffer 1
  • add 1 ml buffer 2 and mix slightly
  • put on ice
  • fill 8 ml buffer 3 into a 14 ml corex centrifugation tube
  • overlay the 8 ml buffer 3 with 1 ml cell suspension so that the tubes are ready for centrifugation 10 min after buffer 2 has been added to the cells
  • centrifuge 8500 rpm 20 min 4°C (ideally in a swing-out rotor)
  • carefully remove supernatant completely
    
• MNase digestion
• resuspend pellet in 1 ml MNase digestion buffer
• aliquot 500 µl of this suspension in 1.5 ml Eppendorf tubes
• add 1 µl MNase (15 U) and incubate 30 sec to 8 min at 37°C

  The digestion time has to be determined experimentally and can be different for each cell type and each MNase preparation. In order to determine the optimal digestion time, transfer every minute 100 µl of the reaction mix into Eppendorf tubes containing 10 µl 0.5 M EDTA on ice. Mix 50 µl of each fraction with 50 µl phenol-chloroform, vortex, centrifuge and apply 20 µl of the upper phase on the 2% SB gel. Run the gel (135V, 25 min). Optimal digestion is achieved when DNA fragments corresponding to 1 - 5 nucleosomes are visible.

  
  

  typical incubation times
  MEL SK23	2 min
  BT20	2 min
  BT474	2 min 30 sec
  GM10323	2 min 30 sec
  human fibroblasts	3 min
  OPM-2	2...6 min
  RPMI	2...6 min

• to stop the reaction add 20 µl 0.5 M EDTA to each 500 µl MNase digest and put the tube on ice
• centrifuge 13000 rpm 10 min 4°C
• transfer the supernatant to a new tube
• centrifuge 13000 rpm 10 min 4°C (this step eliminates the background noise of unspecific enrichment of chromatin), repeat this centrifigation step
• transfer the supernatant to a new tube (S1)
  • it can be necessary to remove chromatin from the pellet by dialysis, in this case prepare 20 ml dialysis buffer (1mM Tris/Cl, 200 µM EDTA, 200 µM PMSF, 5 mM sodium butyrate, resuspend the pellet P1 in 550 µl dialysis buffer and dialyze overnight at 4°C)
  • centrifuge 13000 rpm 10 min 4°C
  • supernatant is fraction S2
• use 50 µl of S1 and S2 for phenol/chlorofrom extraction, centrifuge and load 20 µl of supernatant on 2% SB gel (135V, 25 min)
• dilute 5µl of S1 1/20 with water and quantify chromatin by measuring OD at 260 nm against 5µl of MNase digestion buffer in water (In general we find 40-100 µg/ml DNA in the undiluted S1, OD260/280 values can be bad because there is a lot of protein in the solution. DNA quantification is therefore not precise but sufficient for reproducibility.)
  
• incubation with antibody
  • Ideally, the antibody should be in excess over the protein you want to precipitate. The antigen/antibody ration must be determined experimentally for each antibody . Prepare a dilution series of your chromatin in MNase buffer starting with 20 - 40 µg DNA for histon ChIP.
  • Add appropriate amounts of stock solutions to generate the
    

    antibody incubation buffer
    NaCl	50 mM
    Tris/Cl	20 mM
    sodium butyrate	20 mM
    EDTA	5 mM
    PMSF	100 µM

  • dissolve chromatin from S1 (and S2 in you have dialyzed) in 1 ml buffer
  • add 2 µg antibody
  • incubate overnight at 4°C on a rotating wheel

day 2...

• precipitation
  • add 50 µl of protein A - sepharose to each tube
  • incubate at least 4 h at 4°C on a rotating wheel
  • prepare washing buffers (10 ml / tube) and cool down to 4°C:
    

    washing buffers A B C			50 ml	100 ml	200 ml	300 ml
    	Tris/Cl	50 mM	2.5 ml	5 ml	10 ml	15 ml
    	EDTA	10 mM	1 ml	2 ml	4 ml	6 ml
    	sodium butyrate	5 mM	100 µl	200 µl	400 µl	600 µl
    washing buffer A	NaCl	75 mM	750 µl	1.5 ml	3 ml	4.5 ml
    washing buffer B	NaCl	125 mM	1.25 ml	2.5 ml	5 ml	7.5 ml
    washing buffer C	NaCl	175 mM	1.75 ml	3.5 ml	7 ml	10.5 ml

  • centrifuge chromatin/antibody mixture 10 min 4°C 11600 g
  • keep the supernatant in a 2 ml tube. This is the unbound fraction UB.
  • resuspend the pellet in approx. 1 ml washing buffer A and transfer into a 15 ml Falcon tube containing 9 ml washing buffer A
  • mix briefly and centrifuge 10 min 4000 rpm 4°C
  • pour off supernatant
  • add 10 ml washing buffer B, mix briefly and centrifuge 10 min 4000 rpm 4°C
  • pour off supernatant
  • add 10 ml washing buffer C, mix briefly and centrifuge 10 min 4000 rpm 4°C
  • pour off supernatant
  • centrifuge 10 min 4000 rpm 4°C
  • remove remaining supernatant completely
  • resuspend pellet in 500 µl elution buffer
    

    elution buffer		10 ml	20 ml
    SDS (20% stock)	1 %	500 µl	1ml
    Tris/Cl	20 mM	200 µl	400 µl
    NaCl	50 mM	100 µl	200 µl
    EDTA	5 mM	100 µl	200 µl
    sodium butyrate	20 mM	80 µl	160µl
    PMSF	100 µM	40 µl	80 µl
    water		to 10 ml	to 20 ml

  • transfer suspension to a 1.5 ml Eppendorf tube
  • incubate 15 min at RT on a rotating wheel
  • centrifuge 10 min 11600 g 18°C
  • transfer supernatant to a 1.5 ml Eppendorf tube
  • This is the bound fraction B.
    
• DNA extraction
• extract DNA with phenol/chloroform from fractions B and UB
• add 1 µl of a 20 g/l glycogen stock solution
• add NaCl to 250 mM (26 µl and 52 µl) and add 1 volume isopropanol
• put overnight at -20°C
• precipitate by centrifugation and wash with 70% ethanol
• dry the pellet and resuspend in 20 µl 10 mM Tris/Cl or qPCR grade water
• use 1 µl of this DNA for PCR in 25 µl reactions (quantitative real-time PCR) or 10 µl (PCR)

Ref.:

Umlauf D, Goto Y, Feil R. "Site-Specific Analysis of Histone Methylation and Acetylation" Methods Mol Biol. 2004;287:99-120. [PubMed]

O'Neill LP, Turner BM. "Immunoprecipitation of native chromatin: NChIP." Methods. 2003 Sep;31(1):76-82. [PubMed]