11.8  Chromatin Immunoprecipitation

 

Chaoyang Cheng, Naoko Onodera, Tetsuya Kurata

 

 

 

 

角丸四角形吹き出し: Crosslink is efficient reaction while Revers crosslink is hard.Step1 Crosslink Reaction

角丸四角形吹き出し:  


Cross link

 

@ Sample preparation of moss。(0.5-1.5g fresh weight。)

 

A Wash sample by MilliQ H20Cross link in 37ml 1% formaldehyde and vacuum.

   Arabidopsis (Seedling) 9min,

      Moss (Gametophore) 20min,

      Moss (Protonema) 15min

 

B Add 2.5 ml 2MGlycineVacuume 5min to stop fixation.

C Wash sample by milliQ H20 in several times, drain H20 from sample with kimwipe.

 

D Freeze it in LN2Store at -80.

 

 

 

 

 

 

Step2 Chromatin Extraction and Sonication

 

@ Sterilize the funnelMeshmortarpestle by autoclave.

 

A Preparation of Extraction buffer1,2,3 and Ice box.  Set centrifuge at 4.  Preparation of Nuclei lysis buffer, ChIP dilution buffer

 

Stock Reagent

Amount

Extraction buffer1

2M Sucrose

10ml

1 sample 25ml30ml

1M Tris-HCl

500μl

 

1M MgCl2

500μl

 

14.3M β-ME

17.5μl

 

0.2M PMSF

25μl

 

P(Complete tablet)

1 tablet

 

D.W.

up to 50ml

 

 

 

Extraction buffer2

2M Sucrose

625μl

1 sample 1ml

1M Tris-HCl

50μl

 

1M MgCl2

50μl

 

20% TritonX-100

250μl

 

14.3M β-ME

1.75μl

 

0.2M PMSF

2.5μl

 

P(Complete Mini tablet)

1/2 tablet

 

D.W.

up to 5ml

 

 

 

Extraction buffer3

2M Sucrose

4.25ml

1 sample 600μl

1M Tris-HCl

50μl

 

20% TritonX-100

37.5μl

 

1M MgCl2

10μl

 

14.3M β-ME

1.75μl

 

0.2M PMSF

2.5μl

 

P(Complete Mini tablet)

1/2 tablet

 

D.W.

up to 5ml

 

 

 

Nuclei Lysis Buffer

1M Tris-HCl

500μl

1 sample 300μl

0.5M EDTA

200μl

 

20% SDS

500μl

 

P(Complete Mini tablet)

1 tablet

 

D.W.

up to 10ml

 

 

 

ChIP dilution buffer

20% TritonX-100

550μl

1sample1ml

0.5M EDTA

24μl

 

1M Tris-HCl

167μl

 

5M NaCl

334μl

 

D.W.

up to 10ml

 

B Grind -80 stored sample to powder.  Add 30ml Extraction buffer1, Incubate it 5min on ice.

 

CFilter it by φ48Mesh (4-6 sheets)centrifuge 3000g,4,20min.

 

D To ppt, add 1ml Extraction buffer2, mix completely.

 

E Transfer it into new 1.5ml tube, centrifuge 12000g,4,10minAfter centrifuge, ppt should be white-brown color.

 

F To pptAdd 300μl Extraction Buffer3, mix completely.

 

G Prepare 1.5ml tube containing 300μl fresh Extraction Buffer3, pour F-sup onto fresh Extraction Buffer.

 

H Centrifuge 16000g,4,1h.  Add Nuclei lysis buffer,300ul to ppt.

Clitical Step 


I Fragmentation of chromatin.

 

Use Sonicator; 10sec×6 times(Pipetting every cycle and incubate 1min on ice), or CAVARIS for SOLiD-ChIP-seq.

 

 

Step3 Immunoprecipitation

 

antibodyProteinA agarose beads

 

@ Add ChIP dilution buffer into sonication sample up to 1ml.

A Add 40μl ProteinA agarose, 4,1h Rotation.

 

B Centrifuge 12000g,4,30sec. Transfer sup into new tube.

C Add antibody (no antibody for Mock sample) , 4 4hovernight Rotation.

 

四角形吹き出し: For ChIP, polyclonal antibody is better than monoclonal. Since Fixation may mask certain motif.

 

 

 

 

 


Step4 Wash

 

@ Add 50μl ProteinA agarose, 4,1h Rotation.

 

A Preparation of Wash bufferElution Buffer.  Set Incubater at 65.

 

 

Stock Reagent

Amount(5ml)

Amount(15ml)

Amount(25ml)

Low salt wash buffer

5M NaCl

150μl

450μl

750μl

1sample1.3ml×2

20% SDS

25μl

75μl

125μl

 

20% TritonX-100

250μl

750μl

1,25ml

 

0.5M EDTA

20μl

60μl

100μl

 

1M Tris-HCl

100μl

300μl

500μl

 

D.W.

up tp 5 ml

up tp 15 ml

up to 25ml

 

 

 

 

 

High salt wash buffer

5M NaCl

500μl

1.5ml

2,5ml

1sample1.3ml×2

20% SDS

25μl

75μl

125μl

 

20% TritonX-100

250μl

750μl

1,25ml

 

0.5M EDTA

20μl

60μl

100μl

 

1M Tris-HCl

100μl

300μl

500μl

 

D.W.

up tp 5 ml

up tp 15 ml

up to 25ml

 

 

 

 

 

LiCl wash buffer

4M LiCl

312.5μl

937.5μl

1.5625ml

1sample1.3ml×2

20% NP40

250μl

750μl

1.25ml

 

sodium deoxycholate

0.05g

0.15g

0.25g

 

0.5M EDTA

10μl

30μl

50μl

 

1M Tris-HCl

50μl

150μl

250μl

 

D.W.

up tp 5 ml

up tp 15 ml

up to 25ml

 

 

 

 

 

TE buffer

1M Tris-HCl

500μl

1.5ml

2,5ml

1sample1.3ml×2

0.5M EDTA

10μl

30μl

50μl

 

D.W.

up tp 5 ml

up tp 15 ml

up to 25ml

 

 

 

B Centrifuge 3800g,4,30sec.

 

C Add Low salt buffer 1.3ml to pptgently invertcentrifuge 3800g,4,30sec.

 

D Add Low salt buffer 1.3ml to pptgently invert5min agitation

Centrifuge 3800g,4,30sec.

  Same treatment should be done for High salt,LiCl,TE buffer to wash.

  

F After last washadd 250μl Elution buffer and mixinvert every 3min 65until 15min.

 

G Centrifuge 3800g,20,2min. Transfer sup into new tube.

 

  

H Repeat steps「FG」 one morefinal sample volume should be ~500μl.

 

 

Step5 Reverse crosslink Reaction

 

角丸四角形吹き出し: At least 4hr 65℃ incubation is needed.

Reverse-cross link by NaCl and heat

 

@Add 20ul 5MNaCl, incubate 65 6hovernight for reverse crosslink.  For Input and Sonication samples, add Elution buf up to 500μlalso add 20ul 5MNaCl, incubate 65 6hovernight.

 

AAdd following sol., incubate 45℃、1h

  0.5MEDTA   10μl     

  1MTris-HCl  20μl     

  10mg/mlProteaseK 2μl     

 

BAdd PC 300μl, voltexCentrifuge 10000rpm,5min.  Divide sup to two tubes, add 2.5 vol 100% EtOH1/10 vol 3M Na-citrate1μl glycogen-80℃ 20min, then centrifuge.  Wshh by 70 EtOHwash, dissolve ppt in TE.

 

CFor Input and Sonication samples, add 5ng/ulRNase 37℃、30min.

 

DFor Sonication sample, check the sonication efficiency by 1.2% AGE.