11.8 Chromatin Immunoprecipitation
Chaoyang
Cheng, Naoko Onodera, Tetsuya Kurata
Step1 Crosslink Reaction
Cross
link
@ Sample
preparation of moss。(0.5-1.5g
fresh weight。)
A Wash sample by MilliQ H20、Cross link in 37ml 1% formaldehyde and vacuum.
Arabidopsis (Seedling) → 9min,
Moss
(Gametophore) → 20min,
Moss
(Protonema) → 15min
B Add 2.5 ml 2MGlycine、Vacuume
5min to stop fixation.
C Wash sample by milliQ H20 in several times, drain H20 from sample with kimwipe.
D Freeze it in LN2、Store at -80℃.
Step2 Chromatin Extraction and
Sonication
@ Sterilize the
funnel、Mesh、mortar、pestle 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 25ml〜30ml |
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 |
1sample約1ml |
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℃,10min(After centrifuge, ppt
should be white-brown color).
F To ppt、Add
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.
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
antibody+ProteinA
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℃ 4h〜overnight
Rotation.
Step4 Wash
@ Add 50μl ProteinA
agarose, 4℃,1h Rotation.
A Preparation of Wash
buffer、Elution
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 ppt、gently invert、centrifuge 3800g,4℃,30sec.
D Add Low salt
buffer 1.3ml to ppt、gently invert、5min agitation
Centrifuge 3800g,4℃,30sec.
Same treatment should be done for High salt,LiCl,TE buffer to wash.
F After last wash、add 250μl Elution buffer and mix、invert every 3min 65℃until 15min.
G Centrifuge 3800g,20℃,2min. Transfer sup
into new tube.
H Repeat steps「FG」 one more、final sample volume
should be ~500μl.
Step5 Reverse crosslink
Reaction
Reverse-cross
link by NaCl and heat
@Add 20ul 5MNaCl,
incubate 65℃
6h〜overnight for reverse
crosslink. For Input and Sonication samples, add Elution buf
up to 500μl、also add 20ul 5MNaCl,
incubate 65℃ 6h〜overnight.
AAdd following sol., incubate 45℃、1h
0.5MEDTA
10μl
1MTris-HCl
20μl
10mg/mlProteaseK 2μl
BAdd PC 300μl, voltex。Centrifuge 10000rpm,5min. Divide sup to
two tubes, add 2.5 vol 100% EtOH、1/10 vol
3M Na-citrate、1μl glycogen、-80℃ 20min, then centrifuge. Wshh by 70% EtOHでwash, 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.