Griffin:Chromatin Immunoprecipitation

Crosslinking
start with 9 100mm dishes of βTC-3 cells; can use more or less cells, but stay with multiples of three plates


 * Remove media & wash monolayers 2x with 1x PBS.
 * Add 5ml 1% formaldehyde in DMEM (no serum); formaldehyde stock is 37% & incubate 5 minutes @ room temperature.

NOTE: if using a different cell line, it may be necessary to optimize the crosslinking time


 * Add 250μl 2.5M glycine (this stops the crosslinking reaction); incubate 2 minutes @ room temperature.
 * Remove media & wash cells with cold PBS (high temperatures will reverse the formaldehyde crosslinking).
 * Scrape cells with 1.4ml cold PBS per plate; add 1ml PBS to plate, scrape, and transfer cells; then wash plate with 0.4ml PBS.
 * Transfer cells to eppendorf tubes on ice, spin 5K, 5 minutes to pellet cells & aspirate off supernatant.
 * Resuspend cell pellet in 200μl SDS lysis buffer per tube & incubate 10 minutes on ice

Option A; Virsonic Sonicator

 * Add 250mg glass beads (diameter ≤106μM; Sigma G-4649) to eppendorf tubes; chill on ice.
 * Transfer 3 reactions per tube of glass beads; final volume of 600μl in each tube.
 * Add 6 ul of 0.1 M PMSF to each tube.

Sonicate each tube 8-times, for 10 seconds at power setting 4

spin 10 minutes in cold box at full speed.
 * Take into the cold room: ice bucket with samples (put tubes directly in ice, do not use a metal holder), 15ml conical tube with distilled water, ice bucket with dry ice chunks, ear protection, timer, pencil and paper, set timer to count up.
 * Start sonication of first sample at :00; sonicate for ten seconds, place sample in dry ice, then sonicate water to clean tip.
 * Start second sample at :30, place second sample in dry ice; immediately transfer first sample from dry to wet ice (tubes should be in dry ice for 30 seconds).
 * Repeat 30 second cycles until all samples have been sonicated 10 times - use pencil/paper to mark each round of sonication to help count
 * Transfer supernatants to new tubes; discard beads
 * Aliquot supernatant at 100μl; store @ -80°C
 * To one aliquot (or whatever is left over - it does not have to be 100μl), add 400μl H2O and 20μl 5M NaCl to check the extent of fragmentation

Option B; Bioruptor Sonicator

 * Pool same samples and aliquot equal amounts per tube. Add COLD water with a thin layer of ice up to the blue line indicated in the water bath.
 * Put 1.5ml microfuge tube (with at least 300ul per sample) into the microfuge adaptor. Always put six tubes in the adaptor before using.  Add additional tubes with water in the spaces needed with the same amount as sample.

Settings: You may need to optimize these setting per cell line or tissue samples.


 * Power: High
 * Cycles: 30 seconds on; 30 seconds off (red and green needles set at  0.5 minutes)
 * Timer: 3x 5 minute cycles

NOTE:Replace COLD water between cycles.

Measure extent of fragmentation

 * Aliquot 50ul of each sample into a microfuge tube. add 450μl H2O and 20μl 5M NaCl to check the extent of fragmentation.
 * Incubate @ 65°C, overnight
 * add:

20μl	1.0M Tris, pH 6.5

10μl	0.5M EDTA, pH 8.0

5μl	10mg/ml proteinase K

2μl	10mg/ml RNase A


 * Incubate @ 55°C, 1-2 hours
 * Extract 2X with 1:1 phenol:CHCl3
 * Extract 1X with CHCl3
 * Add 1ml 100% EtOH; spin 30 minutes @ room temperature, full speed, wash pellets with 70% EtOH; vacuum dry, resuspend in 100μl H2O.
 * Measure concentration.
 * Run 2μg from each sonicated sample on a 1% agarose/TAE gel with both 1kb and 100bp DNA ladders

NOTE:Use loading dye with xylene cyanol only - no bromophenol blue

Correctly sonicated DNA should be almost all below 1kb in size with most around 500-700bp. For different cell lines, it may be necessary to adjust the number of sonication cycles in order to get a true estimate of the fragment size range do not overexpose the gel on the UV camera - that tends to make the smear look broader than it actually is.

Option A

 * Start with 100μl aliquots of sonicated DNA; should include at least two control tubes plus one tube for each antibody to be tested.
 * Add 0.9ml cold dilution buffer + 1mM PMSF + 0.01% (1/1000 dilution) protease inhibitor cocktail for mammalian extracts (Sigma P-8340).

Preclear


 * Add 25μl protein A/G - agarose (Santa Cruz sc-2003); incubate @ 4°C, 1 hour.
 * Spin 10K, 30 seconds; do this and all subsequent spins @ 4°C.
 * Transfer supernatants to new tubes; discard agarose beads.

Precleared extracts are now ready for the immunoprecipitation reaction


 * Add primary antibody to be tested - volume or concentration will have to be determined empirically.
 * To control tubes add species-matched normal IgG or no antibody.
 * Incubate @ 4°C, 1 hour.
 * Add 80μl protein A/G-agarose.
 * Incubate @ 4°C, 3 hours.
 * spin 10K, 30 seconds.
 * Save supernatant from no antibody control reaction (to be used as an input control for PCR).
 * Carefully remove and discard supernatant from all reactions, removing as much as possible without disturbing the agarose beads.
 * Wash beads with 1ml of each solution below for 5 minutes @ 4°C (five washes total); spin @ 10K for 30 seconds after each wash to pellet agarose.

Wash 1) low salt bufffer

Wash 2) high salt buffer

Wash 3) LiCl buffer

Wash 4) TE

Wash 5) TE


 * To washed beads, add 0.5ml elution buffer (freshly made) + 20μl 5M NaCl.
 * To 0.5ml of the no antibody supernatant, add 20μl 5M NaCl.
 * Incubate samples and controls @ 65°C, overnight.
 * Spin tubes at 10K for 30 seconds and transfer the supernatant to a new tube.

add:

20μl	1.0M Tris, pH 6.5

10μl	0.5M EDTA, pH 8.0

5μl	10mg/ml proteinase K

2μl	10mg/ml RNase A


 * Incubate @ 55°C, 1-2 hours.
 * Extract 2X with 1:1 phenol:CHCl3.
 * Extract 1X with CHCl3.
 * Add 40μg glycogen azure (Pellet Paint) + 1ml 100% EtOH.
 * Spin 30 minutes @ room temperature, full speed.
 * Wash pellets with 70% EtOH; vacuum dry.
 * Resuspend in 105μl nuclease-free H2O (Promega P119C) @ room temperature, overnight.

Option B

 * Start with 100μl aliquots of sonicated DNA; should include at least two control tubes plus one tube for each antibody to be tested.
 * Add 0.9ml cold dilution buffer + 1mM PMSF + 0.01% (1/1000 dilution) protease inhibitor cocktail for mammalian extracts (Sigma P-8340).

Preclear


 * Add 25μl Protein A Agarose Salmon SpermDNA Upstate (Cat#16-157), incubate @ 4°C, 1 hour.
 * Spin 2K, 1 minute; do this and all subsequent spins @ 4°C.

Precleared extracts are now ready for the immunoprecipitation reaction


 * Transfer supernatants to new tubes; discard agarose beads.
 * Add antibody to be tested - volume or concentration will have to be determined empirically.
 * To control tubes add species-matched normal IgG or no antibody.
 * Incubate @ 4°C, 1 hour.
 * Add 80μl Protein A Agarose Salmon Sperm DNA.
 * Incubate @ 4°C, 3 hours.
 * Spin 2K, 1 minute.


 * Save supernatant from no antibody control reaction (to be used as an input control for PCR).
 * Carefully remove and discard supernatant from all reactions, removing as much as possible without disturbing the agarose beads.
 * Wash beads with 1ml of each solution below for 5 minutes @ 4°C (five washes total); spin @ 10K for 30 seconds after each wash to pellet agarose.

Wash 1) low salt bufffer

Wash 2) high salt buffer

Wash 3) LiCl buffer

Wash 4) TE

Wash 5) TE


 * Freshly prepare elution buffer 0.2% SDS, 0.1M NaHCO3.
 * Elute the histone complex from the antibody by adding 0.250ml elution buffer to pelleted protein A agarose/antibody/histone complex.
 * Incubate 15 minutes @ room temperature, centrifuge 2K, 1 minute.
 * Repeat elution step one time, combine eluates (500ul) and add 20ul 5M NaCl
 * Reverse crosslinks by incubation overnight @ 65°C.


 * Add 20µl Tris-HCl, pH 6.5, 10µl 0.5M EDTA, pH 8.0 + 2µl 10mg/ml Proteinase K and 2µl RNase A 10mg/ml. incubate @ 55°C, 1 to 2 hrs.
 * Phenol/Chloroform extract 2 times, Chloroform extract one time. Add 1µl to 2µl pellet paint Novagen Cat# 69049 + 1ml 100% ETOH.
 * Centrifuge 13K, 30 minute @ room temperature.
 * Wash Pellet with 500µl 70% ETOH.
 * Air Dry pellet and resuspend in 100µl Nuclease-free water.

PCR
Use Ready-to-Go PCR beads in 0.2ml snap-cap tubes (Amersham Pharmacia 27-9556-01)

Set up reactions:

Reaction 1) No added DNA

Reaction 2) 1/100 dilution of the no antibody supernatant

Reaction 3) Eluates from each of the immunoprecipitated samples

To beads, add:


 * H2O to final volume of 25μl
 * 6μl primer set @ 2.5pmol/μl of each oligo
 * 10μl DNA

Reaction conditions: 1. C, 2 minutes

2. Denaturation, 95°C, 30 seconds

3. Annealing Temp, 30 seconds

4. Polymerization, 72°C, 30 seconds

5. Repeat step #2-4, 28-30X

6. 4°C, hold


 * Let machine heat to 95°C before putting samples in block (modified hot start).

Agarose Gel
 * Add 5μl loading dye with xylene cyanol only, no bromophenol blue to each reaction.
 * Run 20μl of each reaction on 1.4% agarose/TAE gel.

Solution Recipes
All recipes are calculated for a final volume of 50ml, except elution buffer.

All solutions should be stored at 4°C, except SDS lysis buffer and elution buffer.

SDS lysis buffer

 * 1%		SDS		(10%)		5.0ml
 * 10mM		EDTA		(0.5M)		1.0ml
 * 50mM		Tris, pH 8 	(1M)		2.5ml

Dilution buffer

 * 0.01%		SDS		(10%)		50μl
 * 1.1%		TritonX-100	(10%)		5.5ml
 * 1.2mM		EDTA		(0.5M)		120μl
 * 16.7mM	Tris, pH 8	(1M)		835μl
 * 167mM	NaCl	(5M)		1.67ml

Low salt buffer

 * 0.1%		SDS		(10%)		0.5ml
 * 1% 		TritonX-100	(10%)		5.0ml
 * 2mM 		EDTA		(0.5M)		0.2ml
 * 20mM 		Tris, pH 8	(1M)		1.0ml
 * 150mM 	NaCl	(5M)		1.5ml

High salt buffer

 * 0.1%		SDS		(10%)		0.5ml
 * 1% 		TritonX-100	(10%)		5.0ml
 * 2mM 		EDTA		(0.5M)		0.2ml
 * 20mM 		Tris, pH 8	(1M)		1.0ml
 * 0.5M	 	NaCl		(5M)		5.0ml

LiCl buffer

 * 0.25M		LiCl		(42.39g/mol)	0.53g
 * 1%		NP-40		(10%)		5ml
 * 1% 		NaDOC		0.5g
 * 1mM		EDTA		(0.5M)		0.1ml
 * 10mM		Tris, pH 8	(1M)		0.5ml

TE

 * 10mM		Tris, pH 8	(1M)		0.5ml
 * 1mM		EDTA		(0.5M)		0.1ml

Elution buffer

 * 0.2%		SDS		(10%)		60μl
 * 0.1M		NaHCO3	(1M)		       0.3ml