Native chromatin immunoprecipitation (N-ChIP) of Schistosoma mansoni (18/02/2014) version 3

This protocol is based on the protocol of David Umlauf.

scheme

Figure 1: General scheme

Before everything begins: prepare solutions...

· 1 M KCl, autoclave

· 5 M NaCl, filter and 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

· 100 mM DTT, 1ml (store at -20°C)

· Roche CompleteProtease Inhibitor (ref: 11 697 498 001)

· 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, sterile filter

· 20 g/l glycogen solution (store at -20°C)

· 2% NaN₂ in water (store at 4°C), NaN₂ is very toxic!

· micro-dialysis units (Slide-a-Lyzer 3500 D cut-off, Pierce 69550). Note: You can also make your own (much cheaper) micro-dialysis units. For details see here (external link, not tested).

preparation of protein A - sepharose

1. weight 250 mg protein A - sepharose in 15 ml falcon tube

2. wash with 10 ml sterile water

3. centrifuge 10 min at 4000 rpm

4. remove supernatant

5. repeat step 3 - 4 four times

6. add sterile water to 5 ml

250 mg Protein A - sepharose swells to approx. 1 ml gel and binds approx. 20 mg human IgG. You will need 50 µl of the protein A sepharose homogeneously mixed in its 5 ml water volume per ChIP. Only if prolonged storage is anticipated (several month) add NaN₂ to 0.02 % and label tube appropriately.

optional if micro dialysis units are not available: preparation of dialysis tubing

1. cut tubing (e.g. VWR international dialysis tube 0.5 mm) into pieces of 10 - 20 cm length

2. boil for 10 min in a large volume of 2% (w/v) sodium bicarbonate and 1 mM EDTA

3. rinse the tubing thoroughly with distilled water

4. boil for 10 min in 1 mM EDTA

5. cool and store in this solution at 4°C

6. before use, wash tubing inside and outside with distilled water

day 1...

· reserve centrifuge and cool down to 4°C

· prepare a 2% 0.5x TBE agarose gel with 20 µl slots

· preheat a water bath to exactly 37°C

· prepare the following solutions with autoclaved distilled water

2x base buffer
6 ml	1 M KCl	60 mM final 1x
0.3 ml	5 M NaCl	15 mM
0.5 ml	1 M MgCl2	5 mM
20 µl	500 mM EDTA	0.1 mM
1.5 ml	1 M Tris/Cl	15 mM
to 50 ml	water
2	Roche protease inhibitor tabletts

buffer1 (0.3 M sucrose)
2.58 g	sucrose
12.5 ml	2x base buffer
50 µl	sodium butyrate
100 µl	PMSF
125 µl	DTT
to 25 ml	water

buffer 2
10 ml	buffer 1 (0.3 M sucrose)
put on 37°C to allow NP40 to be pipetted into the buffer
80 µl	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	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 at 37°C

Dialysis buffer
1mM Tris/Cl, 200 µM EDTA, 200 µM PMSF, 5 mM sodium butyrate
50 µl	Tris/Cl
20 µl	EDTA
400 µl	PMSF
100 µl	sodium butyrate
to 50 ml	water

· put all buffer solutions on ice (except MNase buffer)

· cell lysis

o aliquote 1500 sporocysts, miracidia or 10-20 adults (stored at -80°C or in liquid nitrogen)

o adults:

§ remove excess liquid (if any) and resuspend in 1 ml buffer 1, add 1 ml buffer 2 (lysis buffer) and transfer to Dounce

§ homogenize for 3 min with Dounce (pestle A) on ice

§ put on ice 7 min

o sporocysts or miracidia:

§ centrifuge into Eppendorf tubes, rinse storage tubes with PBS to recover all larvae

§ centrifuge at 3400G, 10 min, 4°C

§ remove supernatant

§ resuspend completely in 1 ml buffer 1

§ do not add human lymphoblast cells as carrier

§ add 1 ml buffer 2 (lysis buffer) and homogenize for 3 min with Dounce (pestle A) on ice

§ put on ice 7 min

o fill 8 ml buffer 3 into a 50 ml corex centrifugation tube

o overlay the 8 ml buffer 3 with 1 ml cell suspension so that the tubes are ready for centrifugation 15 min (sporocysts) of 10 min (adults) after buffer 2 has been added to the cells

o disturb a little bit the interface

o use 2 corex tubes for the sporocysts sample that is in 2 ml buffer 1+2

o mark tubes at the exterior side (to know where to look for the nuclei)

o centrifuge 7800G 20 min 4°C

o carefully remove supernatant completely (by pipetting with 10 ml pipette and then, pipetting with micropipettes 1000µl>100µl>10µl)

· MNase digestion

o resuspend pellet in 1 ml MNase digestion buffer (2 ml for 10 000 miracidia or cercariae)

o aliquot 500 µl of this suspension in 1.5 ml Eppendorf tubes

o add 1 µl MNase (15 U) and incubate 4 min at 37°C

o to stop the reaction add 20 µl 0.5 M EDTA to each 500 µl MNase digest and put the tube on ice

o centrifuge 13000 g 10 min 4°C

o transfer the supernatant to a new tube (S1) and keep the pellet (P1)

o store S1 at -20°C

o Quantify chromatin in S1 with the Qubit® 2.0 Fluorometer (HS DNA assay) following manufacturer instructions.

· Dialysis of P1

o humidify Slide-a-Lyzer with 50µl dialysis buffer

o resuspend the pellet P1 in 100 µl dialysis buffer and dialyze overnight at 4°C against 50 ml dialysis buffer with gentle stirring

day 2...

o the next day, transfer dialysed sample to Eppendorf tubes and...

§ centrifuge 13000 g 10 min 4°C

§ transfer the supernatant to a new tube and repeat the centrifugation 2 times

§ supernatant is fraction S2

o yesterdays supernatant S1...

§ in parallel with the dialyses sample, centrifuge 13000 g 10 min 4°C

§ transfer the supernatant into a new tube and repeat this centrifugation 2 times

o these triple centrifugations are IMPORTANT! They reduce the unspecific background!

o use 50 µl of S1 and S2 for phenol/chlorofrom extraction, centrifuge and load 20 µl of supernatant on 2% 0.5x TBE gel (100V, 25 min)

· incubation with antibody

o 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.

o Add appropriate amounts of stock solutions to generate the

antibody incubation buffer
NaCl	150 mM
Tris/Cl	20 mM
sodium butyrate	20 mM
EDTA	5 mM
PMSF	100 µM

o you can download an Excel worksheet for calculation here (v0.1) or here (v1.0)

o dissolve chromatin from S1 (and S2 if you have dialyzed) in 1 ml buffer

o add about 2 µg antibody

o incubate overnight at 4°C on a rotating wheel

day 3...

· precipitation

o prepare 50 µl of protein A - sepharose for each tube

o wash the beads with milliQ water in 1.5ml Eppendorf: centrifuge 4000G, 5 minutes, remove supernatant and replace with equal volume of sterile water

o repeat the washing two more times

o add 50 µl of protein A - sepharose to each tube

o incubate at least 4 h at 4°C on a rotating wheel

o 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

o centrifuge chromatin/antibody mixture 10 min 4°C 11600 g

o keep the supernatant in a 2 ml tube. This is the unbound fraction UB.

o resuspend the pellet in approx. 1 ml washing buffer A and transfer into a 15 ml Falcon tube containing 9 ml washing buffer A

o mix for 10 min on a rotating wheel at 4°C (speed 6)

o centrifuge 10 min 3400 g 4°C and pour off supernatant (by inversion)

o add 10 ml washing buffer B, mix for 10 min on a rotating wheel at 4°C and centrifuge 10 min 4000 rpm 4°C

o pour off supernatant (by inversion)

o add 10 ml washing buffer C, mix for 10 min on a rotating wheel at 4°C and centrifuge 10 min 4000 rpm 4°C

o pour off supernatant (by inversion)

o centrifuge 10 min 4000 rpm 4°C

o remove remaining supernatant completely (not by inversion but by pipetting with micropipettes 1000 µl, then 100 µl, then 10µl)

o 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

o transfer suspension to a 1.5 ml Eppendorf tube

o incubate 15 min at RT on a rotating wheel

o centrifuge 10 min 11600 g 18°C

o transfer supernatant to a 1.5 ml Eppendorf tube

o This is the bound fraction B.

· DNA extraction

o DNA purification with QIAquick PCR Purification Kit (Quiagen, Cat. no. 28104), following manufacturer instructions. Step 3 and 4: Time of centrifugation : 60sec.

o Step 5: Centrifuge the column in a 2 ml collection tube for 1 min, without the cap.

o Step 7: Before the centrifugation, let the column stand for 2 minutes at room temperature. Elution volume: 50 µl.

o In order to obtain more DNA, it is possible to proceed to a second elution in 30 µl

o Quantify purified DNA with the Qubit® 2.0 Fluorometer (HS DNA assay) following manufacturer instructions.

o use 1 µl of this DNA for PCR in 25 µl reactions (quantitative real-time PCR) or 10 µl (PCR)

Ref.:

Cosseau C, Azzi A, Smith K, Freitag M, Mitta G, Grunau C. "Native chromatin immunoprecipitation (N-ChIP) and ChIP-Seq of Schistosoma mansoni: Critical experimental parameters." Mol Biochem Parasitol. 2009;166:70-6. [PubMed]

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]