ChIP-chip v1.0

From Genome@Yale

Immunoprecipitate TAF1 associated chromatin from IMR90 cells

Contents 1 Overview 2 Methods 2.1 Immunoprecipitation 2.1.1 Chromatin Isolation 2.1.2 Preparation of beads 2.1.3 Immunoprecipitation 2.1.4 Washing beads 2.1.5 Elution from beads and reversal of cross links 2.1.6 Linker Preparation 2.1.7 DNA Precipitation 2.2 LM-PCR 2.2.1 Blunt DNA 2.2.2 Ligate DNA 2.2.3 LM-PCR 2.3 Hybridization 2.3.1 Random Priming of LM-PCR product 2.3.2 Array Pre-hybridization 2.3.3 Preparation of Hybridization Solution 2.3.4 Hybridization 2.3.5 Washing 2.3.6 Scanning

Overview

1st day

prepare dynabeads by incubating with antibody

2nd day

prepare chromatin extract
setup chromatin immunoprecipitation

3rd day

wash beads
elution of DNA from beads and reversal of crosslinks
prepare linkers

4th day

proteinase K treatment
phenol, chloroform extractions
RNase A treatment

Qiaquick PCR purification
blunt DNA
extract/precipitate blunted DNA
ligate linkers

5th day

LM-PCR
label ChIP and input DNA
prehybe slides
hybe slides

6th day

wash slides
scan slides

Methods

Immunoprecipitation

1st day

Prepare 5mg/mL BSA in PBS (0.5g BSA in 100mL PBS) immediately before use

1. Add 600uL (for 6 ChIP reactions) of sheep anti-mouse IgG dynabeads (Dynal, catalog number 110.01) to a 15mL conical tube.
2. Spin for 5 minutes at centrifuge 2K rpm 4°C. Remove supernatant with a pipette.
3. Resuspend the dynabeads in 10mL PBS containing 5mg/mL BSA to each tube. Spin for 5 minutes at centrifuge 2K rpm 4°C.
4. Wash 3 times. Resuspend the dynabeads in 1000uL PBS+BSA.
5. Add 300uL (60ug antibody for 6 ChIP reactions) TAF1 antibody (Santa Cruz Biotech, cat# sc-735) to the dynabeads.
6. Incubate overnight on a rotating platform at 4°C.

Chromatin Isolation

2nd day

recommend using a crosslinked cell pellet from 1 billion cells

Solutions

Lysis Buffer 1 (L1). Add 1 protease inhibitor tablet (Roche Complete cat #1 697 498).

Final                  Stock   Volume for 60mL 2X
50mM Hepes pH 7.5      1M      3mL
140mM NaCl             5M      1.68mL
1mM EDTA               0.5M    120uL
10% Glycerol           100%    6mL
0.5% NP-40             10%     3mL
0.25% Triton X-100     10%     0.15mL
dH2O                   100%    46.05mL

Buffer 2 (L2, Protein Extraction Buffer) Kept at RT. Add 1 protease inhibitor tablet per 50mL.

Final            Stock	  Volume for 50mL 2X
200mM NaCl       5M      2mL
1mM EDTA pH 8    0.5M    100uL
0.5mM EGTA       0.5M    50uL
10mM Tris, pH 8  1M      500uL
dH2O             100%    47.35mL

Buffer 3 (L3, Chromatin Extraction Buffer) Kept on ICE. Add 1 protease inhibitor tablets per 50mL.

Final                  Stock   10mL    Vol for 20mL
1mM EDTA pH 8          0.5M    20uL    40uL
0.5mM EGTA pH 8        0.5M    10uL    20uL
10mM Tris pH 8         1M      100uL   200uL
Complete (Roche)       50X     200uL   400uL
dH2O                   100%    9.67mL  19.34mL

1. Thaw cell pellets in ice water (takes 1hour or longer). Resuspend cells in 10mL of buffer L1. Rock at 4°C for 10 minutes.
2. Spin at 3000 rpm in 10 minutes at 4°C.
3. Resuspend each tube of cells in 10mL of buffer L2.
4. Rock at room temperature for 10 minutes.
5. Pellet nuclei in a tabletop centrifuge by spinning at 3K rpm, 10 minutes at 4°C.
6. Resuspend nuclei in 5mL buffer L3. Transfer to a 15mL conical tube.
7. Sonicate the cell suspension with a microtip attached to Branson 450 sonifier, setting at 4 (about 15-20% power output), constant power (place the 15mL conical tube in a 50mL conical tube with ice). Sonicate 8 times for 20 seconds, allowing the suspension to cool on ice between pulses for 40 seconds.
8. Spin out debris at 4K rpm, 15 minutes.
9. OD samples
10. Adjust the final glycerol concentration to 10% with 80% glycerol stock solution.
11. Adjust the final DNA concentrations to 2mg/mL by adding L3 buffer.
12. Freeze chromatin at -80°C in 2mg aliquots (1mL).
13. Chromatin samples stored at -80°C.

Preparation of beads

Prepare 10% DOC (0.3g DOC in 3mL dH2O) immediately before use
Prepare 5mg/mL BSA in PBS (0.2g BSA in 40mL PBS) immediately before use

1. Remove supernatant with a pipette and resuspend in 1mL PBS+BSA
2. Wash 3 times.
3. Resuspend antibody beads in 600uL 1X PBS+BSA

Immunoprecipitation

2nd day

STOCK SOLUTIONS

                     [Final]  Volume per 1mL chromatin
10% Triton X-100     1%       130uL
10% DOC              0.1%     13uL
50X Complete         1X       26uL
1X TE                1X       131uL

1. Set up IP reactions with crude extract.
2. Add following additional reagents to bring the volume to 1.3mL with following concentration:
3. Add 100uL of magnetic beads to each tube and incubate at 4°C overnight in a rotating platform.

Washing beads

3rd day

Requires following reagents:

10% DOC (0.5 g sodium deoxycholate, DOC, in 5mL dH2O) prepare immediately before use.
Make RIPA buffer immediately before use.  Add the stock solutions in the order listed.

RIPA - add in order listed

Components             STOCK   10mL 1X   50mL 6X   100mL 12X
50mM Hepes, pH 8       1M      0.5mL     2.5mL     5mL
1mM EDTA               0.5M    20uL      100uL     200uL
1% NP-40               10%     1mL       5mL       10mL
0.7% DOC               10%     0.7mL     3.5mL     7mL
dH2O                   100%    6.955mL   34.775mL  69.55mL
0.5M LiCl              8M      0.625mL   3.125mL   6.25mL
Complete               50X     0.2mL     1mL       2mL

TE

Components             STOCK   50mL
10mM Tris, pH 8        1M      500uL
1mM EDTA               0.5M    100uL
dH2O                   100%    49.4mL

1. Use a magnet MPC-S (Dynal, catalog # 120.20) to precipitate the beads, save the 1st supernatant. Wash 8 times with 1mL RIPA buffer. Remove buffer by aspiration.
2. Wash once with 1mL TE.
3. After removing the TE by aspiration, spin the tubes for 3 minutes at 3000rpm and remove remaining liquid with a pipet.

Elution from beads and reversal of cross links

Elution Buffer (TE+SDS)

Components             STOCK   50mL
10mM Tris, pH 8        1M      500uL
1mM EDTA               0.5M    100uL
1% SDS                 10%     5mL
dH2O                   100%    44.35mL

1. Add 50uL of elution buffer, vortex briefly to resuspend the beads and incubate at 65°C for 10 minutes. Vortex briefly every 2 minutes during incubation.
2. Spin for 30 seconds at maximum speed and transfer liquid to a new tube.
3. Add 120uL Elution Buffer to the supernatant in the new tube. Reverse crosslink at 65°C O/N in incubator. Also reverse crosslink 25uL (50ug or less) input chromatin.

Linker Preparation

Linker Sequences

oJW102 (5’-GCGGTGACCCGGGAGATCTGAATTC-3’, HPLC purified) 
oJW103 (5’-GAATTCAGATC-3’, HPLC purified)

1. oJW102 and oJW103 oligos are dissolved in dH2O at 40 uM concentration.
2. To 250uL 1M Tris pH 7.9, add 375uL 40uM oligo oJW102 and 375uL 40uM oligo oJW103.
3. Aliquot in 100uL volumes in Eppendorf tubes.
4. Place the tubes in 95°C heat block for 5 minutes.
5. Transfer samples to 70°C heat block 15 minutes.
6. Remove the block from the heater and place it at room temperature and allow it to cool to 25°C.
7. Transfer the block to 4°C and incubate overnight.
8. Store at -20°C.

DNA Precipitation

3rd day

Proteinase K Mix

1X Sample                    10X  	13X
140uL TE                     1.4mL     1.820mL
3uL Glycogen 20mg/mL         30uL      39uL
7uL Proteinase K 20mg/mL     70uL      91uL

1. Add 150uL Proteinase K Mix to each tube. Vortex.
2. Incubate for 2 hours at 37°C.
3. Add NaCl to 200mM final (13uL 5M).
4. Extract 2X with 300uL phenol.
5. Extract once with 300uL chloroform.
6. Add 700uL 100% EtOH, vortex.
7. Incubate -80°C 15-30 minutes.
8. Spin at 14K rpm for 15 minutes at 4°C
9. Wash pellet with 500uL cold 80% EtOH, vortex, spin 5 minutes at 4°C at 14K rpm.
10. Air dry pellet 5 minutes and resuspend in 50uL TE containing 10ug RNase A (20uL 10mg/mL, RNaseA in 1000uL TE).
11. Incubate 2 hour at 37°C
12. Qiagen PCR purify DNA and elute in 50uL.
13. OD input DNA samples for input chromatin samples only (ChIP DNA concentration is too low to be OD’ed).
14. Prepare 50uL of diluted input DNA sample at concentration of 20ng/uL.

LM-PCR

Blunt DNA

This step is performed on the PCR machine.

Blunting Mix

Components             1X      14X       18X
10X NEB #2 (blue)      11uL    154uL     198uL
10mg/mL BSA NEB        0.5uL   7uL       9uL
20mM dNTP              0.5uL   7uL       9uL
T4 DNA pol 3U/uL       0.2uL   2.8uL     3.6uL
Total                  12.2uL  170.8uL   219.6uL

NaOAc/glycogen mix

Components             1X      14X       18X
3M NaOAc               11uL    154uL     198uL
20mg/mL Glycogen       1.0uL   14uL      18uL
Total                  12uL    168uL     216uL

1. In 200uL PCR tubes, add 40uL ChIP DNA or 20ng (1uL) input DNA and bring the total volume to 100uL with dH2O. Store the remaining DNA at -20°C.
2. Add 12.2 uL of Blunting Mix
3. Mix by pipetting and incubate at 12°C for 20 minutes in a PCR machine (Program: \\MAIN\12/20
4. Transfer blunted DNA to new 1.5mL tubes and place on ice.
5. Add 12uL of NaOAc/glycogen mix. Vortex.
6. Add 120uL of phenol/chloroform/isoamyl alcohol (25:24:1, Sigma P-3803). Transfer to a new tube.
7. Vortex and spin 5 minutes at maximum speed.
8. Transfer 110uL to a new 1.5 mL Eppendorf tube and add 230 uL cold EtOH (100%). Vortex. Store at -80°C for 15-30 minutes. Spin for 14K rpm 15 minutes at 4°C.
9. Remove supernatant and wash the pellet with 500uL cold 80% EtOH.
10. Spin for 5 minutes at 4°C.
11. Pipet off supernatant, spin briefly and remove any remaining liquid with pipette. Allow the pellet to dry briefly (5 minutes).
12. Resuspend pellet in 25uL dH20 and place on ice.

Ligate DNA

Ligase Mix

Components                    1X      14X
5X Ligase Buffer              10uL    140uL
15uM annealed linkers         6.7uL   93.8uL
T4 DNA ligase NEB #202L       0.5uL   7uL
dH2O                          8uL     112uL
Total                         25.2uL  352.8uL

1. Add 25uL of cold ligase mix to each tube:
2. Mix by pipetting and incubate overnight at 16°C.
3. The next day, add 6uL of 3M NaOAc to each tube, and 130uL of EtOH. Vortex.
4. Freeze at -80°C for 15-30 minutes, spin 14K for 15minutes.
5. Wash with 500uL 80% EtOH, then spin, air dry 5 minutes.
6. Resuspend the pellet in 25uL of dH2O and transfer to 200uL PCR tubes.

LM-PCR

4th day

PCR Labeling Mix

Components              1X      14X
10X ThermoPol Buffer    4uL     56uL
2.5mM dNTP              5uL     70uL
40uM oJW102             1.25uL  17.5uL
dH2O                    4.75uL  66.5uL
Total                   15uL    210uL

Polymerase Mix

Components              1X      14X
10X ThermoPol Buffer    1uL     14uL
Taq Polymerase 5U/uL    1uL     14uL
PFU Turbo 2.5U/uL       0.01uL  0.14uL
dH2O                    8uL     112uL
Total                   10uL    140uL

1. Place ligated DNA on ice.
2. Add 15uL of PCR labeling mix to tube.
3. Add 10uL of polymerase mix and mix well by pipeting.
4. Transfer to PCR tubes on ice, place in PCR machine and start program (program file “LM-23” within “Mike” folder on the right PCR machine):
5. Purify with Qiaquick PCR purification kit. Elute in 50uL elution buffer. OD samples.

step 1: 55°C for 2';
step 2: 72°C for 5';
step 3: 95°C for 2';
step 4: 95°C for 1';
step 5: 60°C for 1';
step 6: 72°C for 2'; 
step 7: go to step 4 for 22 times;
step 8: 72°C for 5';
step 9: 4°C forever;

Hybridization

Random Priming of LM-PCR product

5th day

Requires following reagents:

BioPrime DNA labeling system (Invitrogen).
DNA made from LM-PCR step.
dNTP “low C” mix: 1.2mM dATP, dTTP, dGTP, each, and 0.6mM dCTP. (Amersham)
Cy3-dCTP & Cy5-dCTP from Amesham.

1. Add 200ng of DNA from the LM-PCR step to tube. Fill the total volume to 22.5uL.
2. Add 20uL of 2.5X random primer solution (from the kit) to tube.
3. Boil for 5 min at 100°C heat block, then put on ice, incubate for 5 minutes.
4. Add 5uL of 10X dNTP low C mix.
5. Add 1.5uL of Cy5-dCTP to IP sample tubes and 1.5uL of Cy3-dCTP to input sample tubes.
6. Add 1uL of high concentration Klenow (Invitrogen). Vortex and spin.
7. Incubate at 37°C for 1.5 hours.
8. Use Qiagen purification kit to purify the DNA. Elute in 50uL. OD DNA.

Array Pre-hybridization

Requires following reagents:

hyb buffer:
70% formamide/3XSSC/14.3% dextran sulfate (made fresh)
  0.143g dextran sulfate, 150uL 20X SSC, 700uL formamide, 75uL dH20
  vortex 15 seconds and rotate for 1 hour)
2.2X SSC/0.22% SDS (made fresh; 110uL 20X SSC, 868uL dH2O, 22uL 10% SDS)
2% of BSA: 1 g of BSA in 50 ml of dH2O
pre-hybridization solution: 2XSSC/0.05%SDS/0.2%BSA
LM-PCR DNA: purified by QiaQuick from random labeling step.
cot-1 DNA (from Invitrogen)

pre-hybridization solution

Components     	100mL	200mL	300mL
20X SSC        	10mL	20mL	30mL
10% SDS        	0.5mL	1mL	1.5mL
2% BSA         	10mL	20mL	30mL
dH2O           	79.5mL	159mL	238.5mL

1. Fresh made pre-hybridization solution: 2X SSC/0.05% SDS/0.2% BSA (use fresh made BSA).
2. Add 80 ml of pre-hybridization solution into a Coplin jar and warm up solution to 42°C in water bath (take about 20-30 min).
3. Soak slides in the Coplin jar (maximum 4 slides/jar) and incubate for 40 min, at 42°C.
4. Rinse with RO water for 10 seconds. Spin at 2K rpm for 30 seconds.

Preparation of Hybridization Solution

1. Mix 2ug Cy3 labeled DNA with 2ug of Cy5 labeled DNA
2. Add 36ug of human Cot-1 DNA and dH2O to a final volume of 130uL.
3. Add 14uL 3M NaOAc, 280uL EtOH. Mix by vortexing, -80°C for 15’, then spin 15 minutes at 14K in cold room.
4. Wash 1X 70% EtOH and air dry (pipet in 200uL and draw it back up and short spin and remove all trace of EtOH).
5. Dissolve the pellet in 22.4uL of buffer (2.2X SSC, 0.22% SDS). Incubate at 37°C for 10 minutes to dissolve.
6. Vortex lightly. Add 20uL Hybridization buffer. Vortex lightly, but thoroughly.
7. Put cap-locks on tubes. Denature probe at 95°C for 5 min, then spin for 30 seconds
8. Incubate at 42°C for 2 minutes.
9. Add 4uL yeast tRNA (10ug/uL).
10. Add 3.0uL of 2% BSA. Vortex lightly but thoroughly. Incubate at 42°C for 5 minutes. Spin briefly.

Hybridization

1. Spot 47uL (in 3 drops) to the array at the edge opposite from the label (bar code). Place cover slip by first touching the edge and dropping the cover slip onto the slide.
2. Add 15uL dH2O to holes on the either side of the slide. Clamp the hybridization chamber.
3. Incubate at 60°C (submerged in 60°C water bath) for overnight (≥16 hours).

Washing

4th day

Prepare following solutions

400ml 2X SSC, 0.1% SDS preheated to 60°C
300ml 0.1X SSC, 0.1% SDS
1L 0.1X SSC

Note: Although the 2X SSC solutions are preheated to 60°C the washes are actually done at room temperature.

1. Fill a Coplin jar with 50ml 2X SSC, 0.1% SDS pre-heated to 60°C. Gently place the array into the jar, barcode up. Let the Gene Array sit for 1 minute then remove each array slowly from the Coplin jar leaving the cover slip behind.
2. Place the array into a slide rack submersed in 2X SSC, 0.1% SDS pre-heated to 60°C in a Wheaton glass staining dish. Incubate the array for 5 minutes with gentle agitation (rocking on the rocker platform).
3. Remove the slide rack from the 2X SSC, 0.1% SDS solution and place in 250ml of room temperature 0.1X SSC, 0.1% SDS in a Wheaton glass staining dish. Incubate for 10 minutes with gentle agitation.
4. Remove the slide rack from the 0.1X SSC, 0.1% SDS solution and place in 250ml of room temperature 0.1X SSC in a Wheaton glass staining dish for 15 seconds. Agitate gently by hand. This quick wash removes much of the residual SDS.
5. Remove the slide rack from the 250mL of 0.1X SSC solution and place it in 250ml of 0.1X SSC in a glass staining dish. Incubate for 1 minute (with gentle agitation if desired).
6. Repeat step 5 two more times (3 total)
7. After 15 seconds remove the array slowly from the slide rack and place into the rack on centrifuge. Balance well. Spin for 1 minute at 1500rpm in eppendorf 5840. After spin, store slides in slide boxes. Protect slides from dust and light until scanned.

Scanning

1. Slides are scanned immediately after washing and drying.
2. Scan slides using Axon 4000B scanner at appropriate resolution (5um for >20K feature arrays) using GenePix scanning software.
3. Adjust PMT gain for each channel so that flourescence from each channel is equivalent.
4. Extract intensities values from each spot using an appropriate gene array list (gal) file.
5. Save the scanned image (.TIFF), image settings (.gps), and the results file (gpr).

Data analysis section describes the standard method for analysis of ChIP-on-chip data.