剑网三阿越藏剑教学:(ChIPs) Protocol for Tissues

Farnham Lab Chromatin Immunoprecipitation (ChIPs) Protocol for Tissues (2006 Revision)

Day 0

Block Staph A cells (Pansorbin, CalBiochem 507862)

1. Thaw 1 tube (200 μL) of Staph A cells for approximately 5 experimental ChIPs.
2. Add 25 μL of herring sperm DNA (Promega D1815), 10 mg/ml); previously boiled for 5 minutes then chilled in ice.
3. Add 25 μL of BSA (NEB B9001S; 10 mg/ml).
4. Incubate on the rotating platform at 4 ^oC overnight.
5. Microfuge for 5 minutes at 14,000 rpm at 4 ^oC.
6. Remove supernatant and wash pellet twice with 1.4 mL dialysis buffer.
7. Resuspend cells in a volume of dialysis buffer without sarkosyl equal to the original starting volume (200 μL for 1 tube). Add PMSF at a 1:100 dilution (see below).

Day 1

We generally use 30 mg of tissue per antibody in every ChIP. The exact amount of tissue depends upon how abundant the protein of interest is, how strongly the antibody binds and how efficient the crosslinking is.

1. Weight frozen or fresh tissue.
2. Chop tissue into small pieces using 2 razor blades (between 1-3 mm3).
3. Transfer tissue into a tube with a screw cap lid and add 10 mL PBS (plus protease inhibitors) per gram of tissue.
4. Add formaldehyde to a final concentration of 1% and rotate tube at room temperature for 15 minutes.
5. Stop the crosslinking reaction by adding fresh glycine to a final concentration of 0.125 M. Continue to rotate at room temp for 5 minutes.
6. Centrifuge tissue samples at low speed (100g or 707 rpm) at 4 ⁰C.
7. Aspirate media and wash once with cold PBS (plus protease inhibitors). Centrifuge.
8. Resuspend tissue in 10 mL cold PBS (plus protease inhibitors) per gram of starting material. Put on ice.

Tissue disaggregation For this step, we use a Medimachine from Becton Dickinson to achieve a single cell suspension. Use 2 medicones (50 μm) per gram of tissue to process.

1. Cut a 1000 μL pipette tip to make the orifice larger.
2. Put between 50-100 mg of tissue resuspended in 1 mL of PBS in each medicone.
3. Grind tissue for 2 minutes.
4. Collect cells from medicone by inserting an 18g blunt needle and a 1 mL syringe. Put them on ice.
5. Keep putting between 50-100 mg of tissue resuspended in 1 mL of PBS in each medicone until all the tissue is processed.
6. If necessary, add more PBS (plus protease inhibitors) to tissue for a homogeneous suspension.
7. Check cell suspension by microscope.
8. Centrifuge cells at 1000 rpm at 4 ^oC. Measure cell pellet volume.

Chromatin Preparation

1. Resuspend cell pellet in 6X volume of cell lysis buffer plus protease inhibitors PMSF (10 μL/ mL), aprotinin (1 μL/ mL) and leupeptin (1 μL/ mL). The final volume of cell lysis buffer should be sufficient so that there are no clumps of cells. Incubate on ice for 10-15 minutes.
2. Lyse cells using a B dounce several times to aid in nuclei release.
3. Centrifuge nuclei at 1000g (2235 rpm) at 4 ^oC. Discard supernatant.
4. Resuspend nuclei in 5X volume of nuclei lysis buffer plus the same protease inhibitors as the cell lysis buffer. Incubate on ice for 20 minutes.
5. Flash freeze and thaw nuclei in liquid nitrogen 2 times to aid in nuclear lysis.
6. Divide the sample material in a suitable volume for sonication. 2 mL maximum volume in a 15 mL Polystyrene Conical tube (FALCON 2099).
7. Sonicate chromatin. Bioruptor maximum power. 30 seconds ON followed by 1 min OFF. Total time = 10 minutes.
8. Chromatin confirmation: Take an aliquot equivalent to 10 mg of starting tissue material (approx. 25 μL). Add H2O up to a 100 μL volume. Add 10 μL NaCl 5M. Boil for 15 minutes. Add 1 μL DNase-free RNase. Incubate 30 minutes at 37 ^oC. Add 1 μL Proteinase K. Incubate 15 minutes at 45 ^oC. Centrifuge at full speed. Collect supernatant and purify chromatin using a QIAquick column (Qiagen cat#28104). Elute in 50 μL. Measure DNA concentration by NanoDrop. Run 2-3 μg of chromatin in gel. Sonicated chromatin should have an average length between 500-1000 bp.
9. While performing step 8, transfer sonicated chromatin from step 7 to 2 mL tubes. Centrifuge at 14,000 rpm for 10 minutes at 4 ^oC. Collect and combine supernatants. Measure volume.
10. Aliquot chromatin in tubes. Put in each tube the equivalent to 200 mg of starting tissue material (approx. 500 μL). Flash freeze in liquid nitrogen. Store at -70 ^oC.

Day 2

1. Thaw one tube of chromatin in the cold room using the rotating wheel.
2. Preclear chromatin by adding 50 μL of blocked Staph A cells.
3. Incubate on a rotating platform at 4 ^oC for 15 minutes, no longer.
4. Microfuge at 14,000 rmp for 10 minutes.
5. Transfer supernatant to a clean tube and measure the volume.
6. Divide chromatin equally among your samples.
7. Be sure to include an IgG sample. You can also include a "mock" sample which contains 1X dialysis buffer instead of chromatin (IgG and mock are critical to control for nonspecific interactions and DNA contamination of IP and wash solutions).
8. Remove 10% of the amount of chromatin used for one IP. This is the Total INPUT DNA. Save it for later.
9. Adjust the final volume of each sample with IP dilution buffer plus protease inhibitors if necessary. Samples volumes should be between 500 and 600 μL.
10. Add 3 μg of antibody to each sample.
11. Incubate on the rotating platform at 4 ^oC overnight. If you are using monoclonal antibodies, the next day you should add 3 μg of an appropriate secondary antibody and incubate for an additional 1 hour.

Day 3

1. Add 20 μL of blocked Staph A cells to each sample.
2. Incubate on the rotating platform at room temp for 15 minutes, no longer.
3. Microfuge samples at 14,000 rpm at 4 ^oC.
4. Discard the supernatant using a pipette.
5. Wash pellets twice with 1.4 mL of 1X dialysis buffer (if using a monoclonal antibody, omit the sarkosyl).
6. Wash four times with 1.4 mL of IP buffer (pH 8.0 for monoclonal antibodies).
7. For each wash, dissolve the pellet in 700 μL of buffer. Use a 200 pipette tip to resuspend the pellet. Then add the remaining 700 μL of buffer. For each wash, incubate samples on a rotating platform for 3 minutes then microfuge at 14,000 rmp for 3 minutes at 4 ^oC. Try to remove as much buffer as possible after each wash without aspirating the Staph A cells using a pipette tip.
8. After the last wash, microfuge at 14,000 rpm for 3 minutes at 4 ^oC and remove the last traces of buffer (be sure to orient the pellets in the microfuge).
9. Elute antibody/protein/DNA complexes by adding 100 μL of IP elution buffer.
10. Shake on vortexer for at least 15 minutes at setting "vortex 3".
11. Microfuge at 14,000 rpm for 5 minutes at 4 ^oC.
12. Transfer supernatants to clean tubes.
13. Repeat elution step and combine both elutions in the same tube.
14. Microfuge samples at 14,000 rpm for 5 minutes at 4 ^oC to remove any traces of Staph A cells. Transfer supernatants to clean tubes.
15. Add 20 μL NaCl 5M to a final concentration of 0.45 M.
16. Boil samples in the water bath for 15 minutes to reverse formaldehyde crosslinks.
17. Purify DNA in a QIAquick column.

Total INPUT DNA

1. Add H2O to each Total INPUT DNA sample to a final volume of 1 mL.
2. Add 100 μL NaCl 5M.
3. Boil samples in the water bath for 15 minutes to reverse formaldehyde crosslinks.
4. Add 2.75 μL of DNase-free RNase. Final concentration of 25 μg enzyme/mL.
5. Incubate 30 minutes at 37 ^oC.
6. Add 3 μL of Proteinase K.
7. Incubate 15 minutes at 45 ^oC.
8. Purify DNA in a QIAquick column.

Solutions
Cell Lysis buffer
5 mM PIPES pH 8.0
85 mM KCL
0.5% Igepal (add fresh)
protease inhibitors

Nuclei Lysis buffer
50 mM Tris-Cl pH 8.1
10 mM EDTA
1% SDS
protease inhibitors

IP Dilution buffer
0.01% SDS
1.1% Trition X 100
1.2 mM EDTA
16.7 mM Tris-Cl pH 8.1
167 mM NaCl

1X Dialysis buffer
2 mM EDTA
50 mM Tris-Cl pH 8.0
0.2 % Sarkosyl (omit for monoclonal antibodies)

IP Wash buffer
100 mM Tris-Cl pH 9.0 (8.0 for monoclonal antibodies)
500 mM LiCl
1% Igepal
1% deoxycholic acid
Elution buffer
50 mM NaHCO3
1% SDS

Protease Inhibitors
100 mM PMSF (Sigma P-7626) in ethanol, use at 1:100
10 mg per ml aprotinin (Sigma A-1153) in 0.01 M HEPES pH 8.0, use at 1:1,000
10 mg per ml leupeptin (Sigma L-2884) in water, use at 1:1,000

Staph A Cells
Resuspend 1 gram of lyophilized Staph A cells (Pansorbin, from CalBiochem #507862) in 10 mL of 1X dialysis buffer.
Centifuge at 10,000 rpm for 5 minutes at 4C. Repeat.
Resuspend in 3 mL of 1X PBS plus 3% SDS and 10% BME.
Boil for 30 minutes.
Centifuge at 10,000 rpm for 5 minutes.
Wash in 1X dialysis buffer and centrifuge at 10,000 rpm for 5 minutes. Repeat.
Resuspend in 4 mL of 1X dialysis buffer.
Divide into 200 μL aliquots, snap freeze and store in liquid nitrogen.