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「�F�G」 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.