【交流】染色体免疫沉淀技术详细步骤-young lab ！！！
丁香园论坛
6127
小弟最近做此实验（ChIP），上网搜了不少的文献，经过比较，这个 young lab 的讲的是最详细的。所以发上来和大家分享，希望做这个实验的战友大家多交流！
下面是具体的步骤，附件里面也有word文档.
I. Formaldehyde cross-linking of cells
Use 5x107 – 1x108 cells (70-80% confluency for adhesion cells of 8-12 15 cm2 plates or 175 cm2 flasks) for each immunoprecipitation.
Adherent cells:
1. Add 1/10 volume of fresh 11% Formaldehyde Solution to plates.
2. Swirl plates briefly and let them sit at room temperature for 10 minutes.
3. Add 1/20 volume of 2.5 M glycine to plates to quench formaldehyde.
4. Rinse cells twice with 5 ml 1x PBS. Harvest cells using silicon scraper.
5. Pool cells in 50 ml conical tubes and spin at 1,350 x g for 5 minutes at 4°C (Sorvall Legend RT centrifuge with swinging bucket rotor). Discard supernatant and resuspend pellet in 10 ml 1x PBS per 108 cells.
6. Transfer 5x107 – 1x108 cells to 15ml conical tube and spin at 1,350 x g for 5 minutes at 4°C (Sorvall Legend RT centrifuge with swinging bucket rotor). Discard supernatant.
7. Flash freeze cells in liquid nitrogen and store pellets at –80°C.
Suspension cells:
1. Add 1/10 volume of fresh 11% Formaldehyde Solution to flasks.
2. Swirl flasks briefly and let them sit at room temperature for 20 minutes.
3. Add 1/20 volume of 2.5 M glycine to flasks to quench formaldehyde.
4. Spin cells at 1,350 x g for 5 minutes at 4°C.
5. Pool cells in required number of 50 ml conical tubes and spin at 1,350 x g for 5 minutes at 4°C (Sorvall Legend RT centrifuge with swinging bucket rotor). Dump supernatant.
6. Resuspend cells in 50 ml 1X PBS, spin at 1,350 x g for 5 minutes at 4°C. Discard supernatant. Repeat once.
7. Resuspend in 10 ml per 108 cells. Aliquot 5x107 – 1x108 cells to 15ml conical tubes and spin 1,350 x g for 5 minutes at 4°C (Sorvall Legend RT centrifuge with swinging bucket rotor). Discard supernatant.
8. Flash freeze cells in liquid nitrogen and store pellets at –80°C.
Formaldehyde Solution
Final Conc. Stock For 50 ml
50 mM 1M Hepes-KOH, pH 7.5 2.5 ml
100 mM 5M NaCl 1.0 ml
1 mM 0.5M EDTA 50.0 µl
0.5 mM 0.5M EGTA 100.0 µl
11% 37% Formaldehyde 14.9 ml
ddH2O 31.5 ml
II. Preblock and binding of antibody to magnetic beads
NOTE: Exact type of Dynal bead (Protein A, Protein G, sheep anti-mouse IgG, sheep anti-rabbit IgG, etc.) depends on antibody being used. You can try other brands/kinds of beads, but we have no information about what changes you would have to make in the protocol or what kinds of effects these changes would have on the results.
1. Add 100 µl Dynal magnetic beads to microfuge tube. Add 1 ml block solution. Set up 1 tube per IP.
2. Collect the beads using magnetic stand. Remove supernatant.
3. Wash beads in 1.5 ml block solution two more times.
4. Resuspend beads in 250 µl block solution and add 10 µg of antibody.
5. Incubate overnight on a rotating platform at 4°C.
6. Next day, wash beads as described above (3 times in 1 ml block solution).
7. Resuspend in 100 µl block solution.
Block Solution
Final Conc. Stock For 100 ml
1x 10x PBS 10.0 ml
0.5% BSA (w/v) BSA 500.0 mg
ddH2O 90.0 ml
100.0 ml
III. Cell Sonication
1. Resuspend each pellet of ~108 cells in 5 ml of LB1. Rock at 4°C for 10 min. Spin at 1,350 x g for 5 minutes at 4°C in a tabletop centrifuge.
2. Resuspend each pellet in 5 ml of LB2. Rock gently at room temperature for 10 min. Pellet nuclei in tabletop centrifuge by spinning at 1,350 x g for 5 minutes at 4°C.
3. Resuspend each pellet in each tube in 3 ml LB3.
4. Transfer cells to a homemade “sonication tube” (cut a polypropylene, 15ml conical tube into two pieces at the 7 ml mark)
5. Sonicate suspension with a microtip attached to Misonix 3000 sonicator. Samples should be kept in an ice water bath during sonication. To decrease foaming, initially set output power to 4 and increase manually to final power (7) during first burst; sonicate 7 cycles of 30 sec ON and 60 sec OFF.
[NOTE: You will probably need to optimize sonication conditions. These are suggested starting parameters. Shearing varies greatly depending on cell type, growth conditions, quantity, volume, crosslinking and equipment. Depending on the precise experiment, we use power settings as high as 9, anywhere from 3 – 12 cycles and variable ratios of time ON and time OFF. In general, we look for the lowest settings that result in sheared DNA that ranges from 100 – 600 bp in size.]
6. Add 300 µl of 10% Triton X-100 to sonicated lysate. Split into two 1.5 ml centrifuge tubes. Spin at 20,000 x g for 10 minutes at 4°C to pellet debris.
7. Combine supernatants from the two 1.5 ml centrifuge tubes in a new15 ml conical tube for immunoprecipitation.
8. Save 50 µl of cell lysate from each sample as WCE DNA. Store at -20°C.
NOTE: Add protease inhibitors (final concentration 1x) to all lysis buffers before use.
(Dissolve one Complete Protease Inhibitor Cocktail Tablet (Roche) in 1 ml H2O to make a 50x solution. Store in aliquots at -20°C.)
Lysis Buffer 1 (LB1)
Final Conc. Stock For 100 ml
50 mM 1M Hepes-KOH, pH 7.5 5.0 ml
140 mM 5M NaCl 2.8 ml
1 mM 0.5M EDTA 0.2 ml
10% 50% glycerol 20.0 ml
0.5% 10% NP-40 5.0 ml
0.25% 10% Triton X-100 2.5 ml
ddH2O 64.5 ml
Lysis Buffer 2 (LB2)
Final Conc. Stock For 100 ml
10 mM Tris-HCl, pH 8.0 1.0 ml
200 mM 5M NaCl 4.0 ml
1 mM 0.5M EDTA 0.2 ml
0.5 mM 0.5M EGTA 0.1 ml
ddH2O 94.7 ml
Lysis Buffer 3 (LB3)
Final Conc. Stock For 100 ml
10 mM Tris-HCl, pH 8.0 1.0 ml
100 mM 5M NaCl 2.0 ml
1 mM 0.5M EDTA 0.2 ml
0.5 mM 0.5M EGTA 0.1 ml
0.1% 10% Na-Deoxycholate 1.0 ml
0.5% 20% N-lauroylsarcosine 2.5 ml
ddH2O 93.2 ml
IV. Chromatin immunoprecipitation
1. Add 100 µl antibody/magnetic bead mix from Part II, Step 7 to cell lysates from Part III, Step 7.
2. Gently mix overnight on rotator or rocker at 4°C.
V. Wash, elution, and cross-link reversal
Steps 1 through 6 should be done in a 4°C cold room.
1. Pre-chill one 1.5 ml microfuge tube for each IP.
2. Transfer half the volume of an IP to a pre-chilled tube.
3. Let tubes sit in magnetic stand to collect the beads. Remove supernatant and add remaining IP. Let tubes sit again in magnetic stand to collect the beads
4. Add 1 ml Wash Buffer (RIPA) to each tube. Remove tubes from magnetic stand and shake or agitate tube gently to resuspend beads. Replace tubes in magnetic stand to collect beads. Remove supernatant. Repeat this wash 3-7 more times.
[NOTE: Exact number of washes depends on quality of antibody and may need to be optimized for each antibody.]
5. Wash once with 1 ml TE that contains 50 mM NaCl.
6. Spin at 960 x g for 3 minutes at 4°C and remove any residual TE buffer.
7. Add 210 µl of elution buffer.
8. Elute at 65°C for 15 minutes. Resuspend beads every 2 minutes with brief vortexing.
9. Spin down beads at 16,000 x g for 1 minute at room temperature.
10. Remove 200 µl of supernatant and transfer to new tube. Reverse crosslink of this IP DNA by incubating at 65°C overnight.
11. Thaw 50 µl of WCE reserved after sonication, add 150 µl (3 volumes) of elution buffer and mix. Reverse crosslink of this WCE DNA by incubating at 65°C overnight.
Wash Buffer (RIPA)
Final Conc. Stock For 250 ml
50 mM 1M Hepes-KOH, pH 7.6 12.5 ml
500 mM 5M LiCl 25.0 ml
1 mM 0.5M EDTA 0.5 ml
1% 10% NP-40 25.0 ml
0.7% 10% Na-Deoxycholate 17.5 ml
ddH2O 169.5 ml
Elution Buffer
Final Conc. Stock For 100 ml
50 mM 1M Tris-HCl, pH 8.0 5.0 ml
10 mM 0.5M EDTA 2.0 ml
1% 10% SDS 10.0 ml
ddH2O 83.0 ml
VI. Digestion of cellular protein and RNA
1. Add 200 µl of TE to each tube of IP and WCE DNA to dilute SDS in elution buffer.
2. Add 8 µl of 10 mg/ml RNaseA (0.2 µg/ml final concentration).
3. Mix and incubate at 37°C for 2 hours.
4. Add 4 µl of 20 mg/ml proteinaseK (0.2 µg/ml final concentration).
5. Mix and incubate at 55°C for 2 hours.
6. Add 400ul phenol:chloroform:isoamyl alcohol (P:C:IA) and separate phases with 2 ml Heavy Phaselock tube (follow instructions provided by Eppendorf). Optional: WCE DNA may remain cloudy. If cloudy, repeat extraction one more time.
7. Transfer aqueous layer to new centrifuge tube containing 16 µl of 5M NaCl (200 mM final concentration) and 1.5 µl of 20 µg/µl glycogen (30 µg total).
8. Add 800 µl EtOH. Incubate for 30 min at -80°C.
9. Spin at 20,000 x g for 10 minutes at 4°C to pellet DNA. Wash pellets with 500 µl of 80% EtOH.
10. Dry pellets and resuspend each in 70 µl of 10mM Tris-HCl, pH 8.0.
11. Save 15 µl of IP sample for future checkpoints or verification.
12. Measure DNA concentration of IP and WCE with NanoDrop (NanoDrop Technologies) and dilute WCE DNA to 100 ng/ul.
VII. T4 Fill-in and blunt-end ligation
A. T4 polymerase blunting of DNA ends
Steps 1 through 6 should be kept on ice.
1. Mix 2 µl (200 ng) WCE DNA and 53 µl ddH2O for each IP.
2. Aliquot 55 µl for each IP sample. Keep all tubes on ice.
3. Make blunting master mix on ice:
Final Conc. Stock 1x Mix
1x 10x T4 DNA polymerase buffer 11.0 µl
5 ug 10 µg/µl BSA (NEB) 0.5 ul
40 nM 10mM each dNTP 1.0 µl
1.5 U 3U/µl T4 DNA polymerase (NEB) 0.5 µl
ddH2O 42.0 µl
55.0 µl
4. Add 55 µl of blunting master mix to all samples.
5. Incubate for 20 minutes at 12°C in thermal cycler.
6. Add 11.5 µl of 3M sodium acetate, pH 8.0 and 0.5 µl of 20 µg/µl glycogen (10 µg total).
7. Transfer reaction to pre-chilled Phaselock tubes and extract 1x with 120 µl P:C:IA (follow instructions provided by Eppendorf).
8. Transfer aqueous layer to new centrifuge tube containing 250 µl EtOH. Incubate for 30 minutes at -80°C.
9. Spin at 20,000 x g for 10 minutes at 4°C to pellet DNA. Wash pellets with 500 µl of 80% EtOH.
10. Dry pellets and resuspend each in 25 µl H2O. Chill on ice.
B. Blunt-end ligation
1. Make ligase master mix on ice (25 µl per reaction):
Final Conc. Stock 1x Mix
1x 5x ligase buffer 10.0 µl
2 µM 15 µM linkers (see Appendix) 6.7 ul
200U 400U/µl T4 DNA ligase (NEB) 0.5 µl
ddH2O 7.8 µl
55.0 µl
2. Add 25 µl mix to 25 µl of sample.
3. Incubate 16 hours in 16°C water bath.
4. Add 6 µl of 3M sodium acetate and 130 µl EtOH. Incubate 30 min at -80°C.
5. Spin at 20,000 x g for 10 minutes at 4°C to pellet DNA. Wash pellets with 500 µl of 80% EtOH.
6. Dry pellets and resuspend each in 25 ul H2O.
VIIIa. Ligation-mediated PCR (small-scale)
NOTE: Three protocols are included for LM-PCR. The first is a small-scale procedure (our standard protocol). The second is a medium-scale procedure. The third is a large-scale procedure used for the whole genome analysis.
1. Mix 25 µl of IP sample and 25 µl WCE sample in PCR tubes.
2. Make two buffer mixes per rxn:
Mix A
Final Conc. Stock 1x Mix
1x 10X Thermopol buffer (NEB) 4.00 µl
250 nM dNTP mix (2.5 mM each) 5.00 µl
1 µM oligo oJW102 (40 µM) 1.25 µl
ddH2O 4.75 µl
15.00 µl
Mix B
Final Conc. Stock 1x Mix
1x 10X Thermopol buffer (NEB) 1.0 µl
2.5 U Taq polymerase (5U/µl) 0.5 µl
ddH2O 8.5 µl
10.0 µl
3. Add 15 ul of Mix A to each sample and run program “CHIPCHIP”:
Step1: 55°C 4 minutes
Step2: 72°C 3 minutes
Step3: 95°C 2 minutes
Step4: 95°C 30 seconds
Step5: 60°C 30 seconds
Step6: 72°C 1 minute
Step7: GOTO Step4 24 times
Step8: 72°C 5 minutes
Step9: 4°C HOLD
4. Midway through Step1, add 10 µl Mix B to each tube to hot start reactions. If necessary, pause program in Step1 so tubes remain at 55°C while adding Mix B.
5. After PCR is completed, make precipitation mix:
Precipitation Mix
Final Conc. Stock 1x Mix
750 mM 7.5 M Ammonium acetate 25.0 µl
90% Ethanol 225.0 µl
250.0 µl
6. Pool samples where appropriate. Add 250 µl precipitation mix per 50 µl of PCR reaction. Incubate 30 min at -80°C.
7. Spin at 20,000 x g for 10 minutes at 4°C to pellet DNA. Wash pellets with 500 µl of 80% EtOH.
8. Dry pellets and resuspend each in 50 µl H2O.
9. Measure DNA concentration with NanoDrop (use 10-fold dilutions, if necessary) and normalize all samples to 500 ng/µl.
VIIIb. Ligation-mediated PCR (medium-scale)
1. Mix 25 µl of IP sample and 25 µl WCE sample in PCR tubes.
2. Make two buffer mixes per rxn:
Mix A
Final Conc. Stock 1x Mix
1x 10X Thermopol buffer (NEB) 4.00 µl
250 nM dNTP mix (2.5 mM each) 5.00 µl
1 µM oligo oJW102 (40 µM) 1.25 µl
ddH2O 4.75 µl
15.00 µl
Mix B
Final Conc. Stock 1x Mix
1x 10X Thermopol buffer (NEB) 1.0 µl
2.5 U Taq polymerase (5U/µl) 0.5 µl
ddH2O 8.5 µl
10.0 µl
3. Add 15 ul of Mix A to each sample and run program “LMPCR1”:
Step1: 55°C 4 minutes
Step2: 72°C 3 minutes
Step3: 95°C 2 minutes
Step4: 95°C 30 seconds
Step5: 60°C 30 seconds
Step6: 72°C 1 minute
Step7: GOTO Step4 14 times
Step8: 72°C 5 minutes
Step9: 4°C HOLD
4. Midway through Step1, add 10 µl Mix B to each tube to hot start reactions. If necessary, pause program in Step1 so tubes remain at 55°C while adding Mix B.
5. Transfer product to 1.5 ml centrifuge tube and add 475 ul ddH20 (total ~525ul).
Partial expansion
The following steps are for medium-scale amplification (16 to 48 reactions) of IP and WCE DNA using template generated from the 15 round PCR described above.
10. Make up PCR mixes:
Final Conc. Stock 1x Mix
1x 10x Thermopol buffer (NEB) 5.00 µl
250 nM dNTP mix (2.5 mM each) 5.00 µl
1 µM oligo oJW102 (40 µM) 1.25 µl
2.5 U Taq polymerase (5U/µl) 0.25 µl
ddH2O 33.50 µl
template DNA 5.00 µl
50.00 µl
11. Aliquot 50 µl of PCR mix to individual PCR tubes.
12. Run program “LMPCR2”:
Step1: 95°C 2 minutes
Step2: 95°C 30 seconds
Step5: 60°C 30 seconds
Step6: 72°C 1 minute
Step7: GOTO Step2 24 times
Step8: 72°C 5 minutes
Step9: 4°C HOLD
13. Make precipitation mix:
Precipitation Mix
Final Conc. Stock 1x Mix
750 mM 7.5 M Ammonium acetate 25.0 µl
90% Ethanol 225.0 µl
250.0 µl
14. Pool samples where appropriate. Add 250 µl precipitation mix per 50 µl of PCR reaction. Incubate 30 min at -80°C.
15. Spin at 20,000 x g for 10 minutes at 4°C to pellet DNA. Wash pellets with 500 µl of 80% EtOH.
16. Dry pellets and resuspend each in 50 µl H2O.
17. Measure DNA concentration with NanoDrop (use 10-fold dilutions, if necessary) and normalize all samples to 500 ng/µl.
VIIIc. Ligation-mediated PCR (Large-scale)
1. Mix 25 µl of IP sample and 25 µl WCE sample in PCR tubes.
2. Make two buffer mixes per rxn:
Mix A
Final Conc. Stock 1x Mix
1x 10X Thermopol buffer (NEB) 4.00 µl
250 nM dNTP mix (2.5 mM each) 5.00 µl
1 µM oligo oJW102 (40 µM) 1.25 µl
ddH2O 4.75 µl
15.00 µl
Mix B
Final Conc. Stock 1x Mix
1x 10X Thermopol buffer (NEB) 1.0 µl
2.5 U Taq polymerase (5U/µl) 0.5 µl
ddH2O 8.5 µl
10.0 µl
3. Add 15 ul of Mix A to each sample and run program “LMPCR1”:
Step1: 55°C 4 minutes
Step2: 72°C 3 minutes
Step3: 95°C 2 minutes
Step4: 95°C 30 seconds
Step5: 60°C 30 seconds
Step6: 72°C 1 minute
Step7: GOTO Step4 14 times
Step8: 72°C 5 minutes
Step9: 4°C HOLD
4. Midway through Step1, add 10 µl Mix B to each tube to hot start reactions. If necessary, pause program in Step1 so tubes remain at 55°C while adding Mix B.
5. Transfer product to 1.5 ml centrifuge tube and add 475 ul ddH20 (total ~525ul).
Full expansion
The following steps are for large-scale amplification (96 reactions each) of IP and WCE DNA using template generated from the 15 round PCR described above.
6. Make up PCR mixes:
Final Conc. Stock 1x Mix For 2x96-well plate
1x 10x Thermopol buffer (NEB) 5.00 µl 1.00 ml
250 nM dNTP mix (2.5 mM each) 5.00 µl 1.00 ml
1 µM oligo oJW102 (40 µM) 1.25 µl 250.00 µl
2.5 U Taq polymerase (5U/µl) 0.25 µl 50.00 µl
ddH2O 33.50 µl 6.75 ml
45.00 µl 9.00 ml
7. Split PCR mix into two 4.5 ml aliquots. Add 500 µl of IP DNA to one and 500 µl of WCE DNA to the other.
8. Aliquot 50 µl of total mix per well for an entire 96-well plate. Use one plate for IP DNA and one plate for WCE DNA.
9. Run program “LMPCR2”:
Step1: 95°C 2 minutes
Step2: 95°C 30 seconds
Step5: 60°C 30 seconds
Step6: 72°C 1 minute
Step7: GOTO Step2 24 times
Step8: 72°C 5 minutes
Step9: 4°C HOLD
9. Pool all wells for IP DNA in a 50 ml conical tube. Add 2.5 ml of 7.5M ammonium acetate and 25 ml of EtOH. Repeat for WCE DNA.
10. Split each precipitate mix into two 30 ml Corex tubes. Cover tubes with parafilm. Incubate for 60 min at -80°C.
11. Place tubes in centrifuge (Sorval SS34 rotor). Use appropriate adaptors. Spin at 12,000 x g for 60 minutes at 4°C.
12. Decant supernatant. Add 1 ml of EtOH and dislodge pellet. If necessary, scrape side of tube and vortex vigorously.
13. Transfer pellet and EtOH to a 1.5 ml microfuge tube and spin at 20,000 x g for 5 minutes at 4°C to pellet DNA.
14. Remove supernatant and add 80% EtOH. Resuspend pellet and then spin at 20,000 x g for 5 minutes at 4°C to pellet DNA.
15. Dry pellets and resuspend each in 500 µl H2O.
16. Measure DNA concentration with NanoDrop (use 10-fold dilutions, if necessary) and normalize all samples to 500 ng/µl.
IX. Cy3/Cy5 labeling of IP/WCE material
This is a random-primed, Klenow-based extension protocol derived from Invitrogen’s CGH kit. Our protocol varies from the instructions provided by Invitrogen in both reaction volume and reagent concentrations. We perform 20 reactions per “30 reaction” Invitrogen kit. A pair of reactions (one for each dye) yields enough material for 1-2 hybridizations. To scale up for more arrays, we increase number—not volume—of individual reactions. For whole genome arrays, we performed reactions in 96-well PCR plates (requires 10 CGH kits and 5 tubes of each Cy dye).
1. Open requisite number of CGH kits and consolidate 2.5x Random Primer Solution, lowT dNTP mix, Klenow, and Stop Buffer.
2. To label both IP and WCE, make priming solution mix for each:
Final Conc. Stock 1x Mix 100x Mix
2 µg LM-PCR product (500 ng/ul) 4.0 µl 400.0 µl
1x 2.5x random primer solution 35.0 µl 3.5 ml
ddH2O 36.0 µl 3.6 ml
75.0 µl 7.5 ml
3. Vortex mixture for 30 seconds.
4. Aliquot 75 µl mix/well.
5. Place plates in thermal cycler preheated to 95°C. After 5 minutes at 95°C, immediately transfer plate to ice-water bath for 5 minutes to flash cool reagents.
6. While reactions are cooling, make up label mix:
(we typically use Cy5 mix with IP DNA and Cy3 with WCE DNA)
Final Conc. Stock 1x Mix 100x Mix
110/55 nM lowT dNTP Mix (1.2/0.6 mM) 8.2 µl 820 µl
17 µM Cy5- or Cy3-dUTP (1 mM) 1.5 µl 150 µl
60 U Klenow (40 U/ul) 1.5 µl 150 µl
ddH2O 1.8 µl 180 µl
13.0 ul 1300 µl
7. Vortex mixture for 30 seconds.
8. Aliquot 13 µl label mix/well. Pipette up and down multiple times to mix reagents.
9. Incubate for 3 hours at 37°C. Keep samples in dark.
10. Add 9 µl stop buffer to each well and mix.
At this point, many different clean-up methods can be used to separate dye and nucleotides from longer DNA fragments. For small-scale cleanup, we use bind and elute columns, either CGH columns (Invitrogen) or PCR cleanup columns (Qiagen) and follow manufacturer’s directions. For large-scale preparations, we have used the following protocol.
11. Transfer labeling reactions to Microcon YM-30 filter plate.
12. Spin at 3,000 x g for 15 minutes at room temperature. Filtration will take longer if centrifuge is cold.
13. Make sure DNA retentate is dry. Resuspend DNA in 60 µl ddH2O by pipetting up and down multiple times. Pool identical wells.
14. Measure DNA concentration and dye incorporation using NanoDrop (each plate should yield ~1mg DNA).
15. Labeled DNA can be frozen at this point. For long-term storage, precipitate DNA and leave in pellet form at -20°C shielded from light.
X. Array Hybridization
Note: Keep samples in dark as much as possible (use foil as necessary)
1. Thaw labeled DNA.
2. Combine IP and WCE DNA and add ddH2O to 120 µl total volume per hybridization. Each hybridization requires 5 – 10 µg each of labeled IP and WCE DNA. Maximize amount of DNA within volume limitations. If labeled DNA solutions are too dilute, the maximum volume acceptable at this point is 166.5 µl. Adjust water in Step 6 accordingly.
3. Make up master mix of control nucleic acids. Note that we use the control oligos at 0.1x and not 1x concentration as suggested by the manufacturer:
Final Conc. Stock 1x Mix
750 ng herring sperm DNA (100 ng/µl) 7.5 µl
40 µg yeast tRNA (8 µg/µl) 5.0 µl
10 µg Human Cot-1 DNA (1 µg/µl) 10.0 µl
0.1x Agilent control oligos (10x) 5.0 µl
27.5 µl
4. Add control mixture to sample and mix thoroughly.
5. Aliquot 147.5 ul of sample/control mix to individual Eppendorf tubes (one tube per array to be hybridized).
6. Make up buffer master mix. Add reagents in the order listed:
Final Conc. Stock 1x Mix
ddH2O 46.5 µl
50 mM Na-MES pH 6.9 (500 mM) 50.0 µl
500 mM NaCl (5 M) 50.0 µl
6 mM EDTA (500 mM) 6.0 µl
0.5% Sarcosine (5% -- ultrapure) 50.0 µl
30 % Formamide (ultrapure) 150.0 µl
352.5 µl
7. Add 352.5 ul room temperature buffer mix to each tube. Samples can be left at room temperature during this step.
8. Heat samples for 3 minutes at 95°C.
9. Transfer tubes to 40°C heat block and incubate for 15 minutes.
10. Spin tubes at 13,000 x g for 45 seconds at room temperature.
11. Assemble arrays as described by manufacturer (Agilent Technologies). Briefly, slides bearing gaskets are placed face-up in a hybridization chamber. Hybridization solution is added to the slide and arrays are then placed face-down on the solution. The chamber is assembled and checked for free rotation of hybridization solution.
12. Incubate at 40°C in rotating oven for 20 hours.
XI. Array Washing
Note: Process one array at a time through Step 3. Steps 1 through 3 should be done quickly. Volumes and times here are for sets of 10 arrays during Steps 4 through 9 and may require optimization if you wish to process more or fewer slides.
1. Disassemble hybridization chambers and transfer sandwiched slides to reservoir containing 1 liter of Wash Buffer I.
2. While submerged, IMMEDIATELY and gently separate gasket slide from array slide. Wash array slide with 2 seconds of gentle agitation.
3. Place array in a metal, 30-slide rack submerged in a second dish of 850 ml of Wash I. Separate slides by 2-4 slots in the rack to allow for proper washing. This high volume will result in some spillage in Step 4, but is required to keep the slide submerged as you place it in the rack.
4. Wash for 5 minutes by placing dish on orbital shaker set at 60 rpm.
5. During the wash, set up two dishes, one containing 650 ml of Wash II and one containing 750 ml of Wash II. The second dish will be placed on a magnetic stir plate. Check that a stir bar will be able to move freely when the rack is placed inside.
6. After Step 4, smoothly and rapidly remove slide rack, dip for 1 second in the dish containing 650 ml of Wash II and transfer to the second dish containing 750 ml of Wash II and a stir bar.
7. Wash for 5 minutes with stirring (typically setting 4 on most stir plates).
8. During the wash, set up two dishes, one containing 650 ml of 100% acetonitrile and one containing 750 of Agilent Wash III (follow manufacturer’s directions for preparation of Wash III).
9. After Step 7, remove rack, dip for 1 second in dish containing acetonitrile and transfer to Wash III. Wash for 30 seconds with stirring (setting 4 on most stir plates).
10. SLOWLY and evenly remove rack and slides from Wash III. It should take approximately 10 seconds to lift rack from solution (the slides should be dry at this point).
11. Scan immediately or store in sealed high N2 environment.
Wash I
Final Conc. Stock For 2000 ml
6x 20x SSPE 600.0 ml
ddH2O 1398.0 ml
0.005% 5% N-lauroylsarcosine (ultrapure) 2.0 ml
2000.0 ml
Wash II
Final Conc. Stock For 2000 ml
0.06x 20x SSPE 6.0 ml
ddH2O 1994.0 ml
2000.0 ml
Wash III
(Purchase 1x solution from Agilent)
Appendix:
Oligos for blunt end ligation:
oJW102: 5’-GCGGTGACCCGGGAGATCTGAATTC
oJW103: 5’-GAATTCAGATC
Preparation of 15 µM Linker Stock:
1. Mix the following:
250 µl Tris-HCl (1M) pH 7.9
375 µl oligo oJW102 (40 µM stock)
375 µl oligo oJW103 (40 µM stock)
NOTE: Order these oligos desiccated, then resuspend in ddH20 to 40 µM.
2. Make 50 or 100 ul aliquots in PCR tubes.
3. Place in a thermal cycler and run program LINKER:
95°C for 5 minutes
70°C for 1 minute
Ramp down to 4°C (0.4°C/min)
4°C HOLD
4. Store linkers at -20 °C.
下面是具体的步骤，附件里面也有word文档.
I. Formaldehyde cross-linking of cells
Use 5x107 – 1x108 cells (70-80% confluency for adhesion cells of 8-12 15 cm2 plates or 175 cm2 flasks) for each immunoprecipitation.
Adherent cells:
1. Add 1/10 volume of fresh 11% Formaldehyde Solution to plates.
2. Swirl plates briefly and let them sit at room temperature for 10 minutes.
3. Add 1/20 volume of 2.5 M glycine to plates to quench formaldehyde.
4. Rinse cells twice with 5 ml 1x PBS. Harvest cells using silicon scraper.
5. Pool cells in 50 ml conical tubes and spin at 1,350 x g for 5 minutes at 4°C (Sorvall Legend RT centrifuge with swinging bucket rotor). Discard supernatant and resuspend pellet in 10 ml 1x PBS per 108 cells.
6. Transfer 5x107 – 1x108 cells to 15ml conical tube and spin at 1,350 x g for 5 minutes at 4°C (Sorvall Legend RT centrifuge with swinging bucket rotor). Discard supernatant.
7. Flash freeze cells in liquid nitrogen and store pellets at –80°C.
Suspension cells:
1. Add 1/10 volume of fresh 11% Formaldehyde Solution to flasks.
2. Swirl flasks briefly and let them sit at room temperature for 20 minutes.
3. Add 1/20 volume of 2.5 M glycine to flasks to quench formaldehyde.
4. Spin cells at 1,350 x g for 5 minutes at 4°C.
5. Pool cells in required number of 50 ml conical tubes and spin at 1,350 x g for 5 minutes at 4°C (Sorvall Legend RT centrifuge with swinging bucket rotor). Dump supernatant.
6. Resuspend cells in 50 ml 1X PBS, spin at 1,350 x g for 5 minutes at 4°C. Discard supernatant. Repeat once.
7. Resuspend in 10 ml per 108 cells. Aliquot 5x107 – 1x108 cells to 15ml conical tubes and spin 1,350 x g for 5 minutes at 4°C (Sorvall Legend RT centrifuge with swinging bucket rotor). Discard supernatant.
8. Flash freeze cells in liquid nitrogen and store pellets at –80°C.
Formaldehyde Solution
Final Conc. Stock For 50 ml
50 mM 1M Hepes-KOH, pH 7.5 2.5 ml
100 mM 5M NaCl 1.0 ml
1 mM 0.5M EDTA 50.0 µl
0.5 mM 0.5M EGTA 100.0 µl
11% 37% Formaldehyde 14.9 ml
ddH2O 31.5 ml
II. Preblock and binding of antibody to magnetic beads
NOTE: Exact type of Dynal bead (Protein A, Protein G, sheep anti-mouse IgG, sheep anti-rabbit IgG, etc.) depends on antibody being used. You can try other brands/kinds of beads, but we have no information about what changes you would have to make in the protocol or what kinds of effects these changes would have on the results.
1. Add 100 µl Dynal magnetic beads to microfuge tube. Add 1 ml block solution. Set up 1 tube per IP.
2. Collect the beads using magnetic stand. Remove supernatant.
3. Wash beads in 1.5 ml block solution two more times.
4. Resuspend beads in 250 µl block solution and add 10 µg of antibody.
5. Incubate overnight on a rotating platform at 4°C.
6. Next day, wash beads as described above (3 times in 1 ml block solution).
7. Resuspend in 100 µl block solution.
Block Solution
Final Conc. Stock For 100 ml
1x 10x PBS 10.0 ml
0.5% BSA (w/v) BSA 500.0 mg
ddH2O 90.0 ml
100.0 ml
III. Cell Sonication
1. Resuspend each pellet of ~108 cells in 5 ml of LB1. Rock at 4°C for 10 min. Spin at 1,350 x g for 5 minutes at 4°C in a tabletop centrifuge.
2. Resuspend each pellet in 5 ml of LB2. Rock gently at room temperature for 10 min. Pellet nuclei in tabletop centrifuge by spinning at 1,350 x g for 5 minutes at 4°C.
3. Resuspend each pellet in each tube in 3 ml LB3.
4. Transfer cells to a homemade “sonication tube” (cut a polypropylene, 15ml conical tube into two pieces at the 7 ml mark)
5. Sonicate suspension with a microtip attached to Misonix 3000 sonicator. Samples should be kept in an ice water bath during sonication. To decrease foaming, initially set output power to 4 and increase manually to final power (7) during first burst; sonicate 7 cycles of 30 sec ON and 60 sec OFF.
[NOTE: You will probably need to optimize sonication conditions. These are suggested starting parameters. Shearing varies greatly depending on cell type, growth conditions, quantity, volume, crosslinking and equipment. Depending on the precise experiment, we use power settings as high as 9, anywhere from 3 – 12 cycles and variable ratios of time ON and time OFF. In general, we look for the lowest settings that result in sheared DNA that ranges from 100 – 600 bp in size.]
6. Add 300 µl of 10% Triton X-100 to sonicated lysate. Split into two 1.5 ml centrifuge tubes. Spin at 20,000 x g for 10 minutes at 4°C to pellet debris.
7. Combine supernatants from the two 1.5 ml centrifuge tubes in a new15 ml conical tube for immunoprecipitation.
8. Save 50 µl of cell lysate from each sample as WCE DNA. Store at -20°C.
NOTE: Add protease inhibitors (final concentration 1x) to all lysis buffers before use.
(Dissolve one Complete Protease Inhibitor Cocktail Tablet (Roche) in 1 ml H2O to make a 50x solution. Store in aliquots at -20°C.)
Lysis Buffer 1 (LB1)
Final Conc. Stock For 100 ml
50 mM 1M Hepes-KOH, pH 7.5 5.0 ml
140 mM 5M NaCl 2.8 ml
1 mM 0.5M EDTA 0.2 ml
10% 50% glycerol 20.0 ml
0.5% 10% NP-40 5.0 ml
0.25% 10% Triton X-100 2.5 ml
ddH2O 64.5 ml
Lysis Buffer 2 (LB2)
Final Conc. Stock For 100 ml
10 mM Tris-HCl, pH 8.0 1.0 ml
200 mM 5M NaCl 4.0 ml
1 mM 0.5M EDTA 0.2 ml
0.5 mM 0.5M EGTA 0.1 ml
ddH2O 94.7 ml
Lysis Buffer 3 (LB3)
Final Conc. Stock For 100 ml
10 mM Tris-HCl, pH 8.0 1.0 ml
100 mM 5M NaCl 2.0 ml
1 mM 0.5M EDTA 0.2 ml
0.5 mM 0.5M EGTA 0.1 ml
0.1% 10% Na-Deoxycholate 1.0 ml
0.5% 20% N-lauroylsarcosine 2.5 ml
ddH2O 93.2 ml
IV. Chromatin immunoprecipitation
1. Add 100 µl antibody/magnetic bead mix from Part II, Step 7 to cell lysates from Part III, Step 7.
2. Gently mix overnight on rotator or rocker at 4°C.
V. Wash, elution, and cross-link reversal
Steps 1 through 6 should be done in a 4°C cold room.
1. Pre-chill one 1.5 ml microfuge tube for each IP.
2. Transfer half the volume of an IP to a pre-chilled tube.
3. Let tubes sit in magnetic stand to collect the beads. Remove supernatant and add remaining IP. Let tubes sit again in magnetic stand to collect the beads
4. Add 1 ml Wash Buffer (RIPA) to each tube. Remove tubes from magnetic stand and shake or agitate tube gently to resuspend beads. Replace tubes in magnetic stand to collect beads. Remove supernatant. Repeat this wash 3-7 more times.
[NOTE: Exact number of washes depends on quality of antibody and may need to be optimized for each antibody.]
5. Wash once with 1 ml TE that contains 50 mM NaCl.
6. Spin at 960 x g for 3 minutes at 4°C and remove any residual TE buffer.
7. Add 210 µl of elution buffer.
8. Elute at 65°C for 15 minutes. Resuspend beads every 2 minutes with brief vortexing.
9. Spin down beads at 16,000 x g for 1 minute at room temperature.
10. Remove 200 µl of supernatant and transfer to new tube. Reverse crosslink of this IP DNA by incubating at 65°C overnight.
11. Thaw 50 µl of WCE reserved after sonication, add 150 µl (3 volumes) of elution buffer and mix. Reverse crosslink of this WCE DNA by incubating at 65°C overnight.
Wash Buffer (RIPA)
Final Conc. Stock For 250 ml
50 mM 1M Hepes-KOH, pH 7.6 12.5 ml
500 mM 5M LiCl 25.0 ml
1 mM 0.5M EDTA 0.5 ml
1% 10% NP-40 25.0 ml
0.7% 10% Na-Deoxycholate 17.5 ml
ddH2O 169.5 ml
Elution Buffer
Final Conc. Stock For 100 ml
50 mM 1M Tris-HCl, pH 8.0 5.0 ml
10 mM 0.5M EDTA 2.0 ml
1% 10% SDS 10.0 ml
ddH2O 83.0 ml
VI. Digestion of cellular protein and RNA
1. Add 200 µl of TE to each tube of IP and WCE DNA to dilute SDS in elution buffer.
2. Add 8 µl of 10 mg/ml RNaseA (0.2 µg/ml final concentration).
3. Mix and incubate at 37°C for 2 hours.
4. Add 4 µl of 20 mg/ml proteinaseK (0.2 µg/ml final concentration).
5. Mix and incubate at 55°C for 2 hours.
6. Add 400ul phenol:chloroform:isoamyl alcohol (P:C:IA) and separate phases with 2 ml Heavy Phaselock tube (follow instructions provided by Eppendorf). Optional: WCE DNA may remain cloudy. If cloudy, repeat extraction one more time.
7. Transfer aqueous layer to new centrifuge tube containing 16 µl of 5M NaCl (200 mM final concentration) and 1.5 µl of 20 µg/µl glycogen (30 µg total).
8. Add 800 µl EtOH. Incubate for 30 min at -80°C.
9. Spin at 20,000 x g for 10 minutes at 4°C to pellet DNA. Wash pellets with 500 µl of 80% EtOH.
10. Dry pellets and resuspend each in 70 µl of 10mM Tris-HCl, pH 8.0.
11. Save 15 µl of IP sample for future checkpoints or verification.
12. Measure DNA concentration of IP and WCE with NanoDrop (NanoDrop Technologies) and dilute WCE DNA to 100 ng/ul.
VII. T4 Fill-in and blunt-end ligation
A. T4 polymerase blunting of DNA ends
Steps 1 through 6 should be kept on ice.
1. Mix 2 µl (200 ng) WCE DNA and 53 µl ddH2O for each IP.
2. Aliquot 55 µl for each IP sample. Keep all tubes on ice.
3. Make blunting master mix on ice:
Final Conc. Stock 1x Mix
1x 10x T4 DNA polymerase buffer 11.0 µl
5 ug 10 µg/µl BSA (NEB) 0.5 ul
40 nM 10mM each dNTP 1.0 µl
1.5 U 3U/µl T4 DNA polymerase (NEB) 0.5 µl
ddH2O 42.0 µl
55.0 µl
4. Add 55 µl of blunting master mix to all samples.
5. Incubate for 20 minutes at 12°C in thermal cycler.
6. Add 11.5 µl of 3M sodium acetate, pH 8.0 and 0.5 µl of 20 µg/µl glycogen (10 µg total).
7. Transfer reaction to pre-chilled Phaselock tubes and extract 1x with 120 µl P:C:IA (follow instructions provided by Eppendorf).
8. Transfer aqueous layer to new centrifuge tube containing 250 µl EtOH. Incubate for 30 minutes at -80°C.
9. Spin at 20,000 x g for 10 minutes at 4°C to pellet DNA. Wash pellets with 500 µl of 80% EtOH.
10. Dry pellets and resuspend each in 25 µl H2O. Chill on ice.
B. Blunt-end ligation
1. Make ligase master mix on ice (25 µl per reaction):
Final Conc. Stock 1x Mix
1x 5x ligase buffer 10.0 µl
2 µM 15 µM linkers (see Appendix) 6.7 ul
200U 400U/µl T4 DNA ligase (NEB) 0.5 µl
ddH2O 7.8 µl
55.0 µl
2. Add 25 µl mix to 25 µl of sample.
3. Incubate 16 hours in 16°C water bath.
4. Add 6 µl of 3M sodium acetate and 130 µl EtOH. Incubate 30 min at -80°C.
5. Spin at 20,000 x g for 10 minutes at 4°C to pellet DNA. Wash pellets with 500 µl of 80% EtOH.
6. Dry pellets and resuspend each in 25 ul H2O.
VIIIa. Ligation-mediated PCR (small-scale)
NOTE: Three protocols are included for LM-PCR. The first is a small-scale procedure (our standard protocol). The second is a medium-scale procedure. The third is a large-scale procedure used for the whole genome analysis.
1. Mix 25 µl of IP sample and 25 µl WCE sample in PCR tubes.
2. Make two buffer mixes per rxn:
Mix A
Final Conc. Stock 1x Mix
1x 10X Thermopol buffer (NEB) 4.00 µl
250 nM dNTP mix (2.5 mM each) 5.00 µl
1 µM oligo oJW102 (40 µM) 1.25 µl
ddH2O 4.75 µl
15.00 µl
Mix B
Final Conc. Stock 1x Mix
1x 10X Thermopol buffer (NEB) 1.0 µl
2.5 U Taq polymerase (5U/µl) 0.5 µl
ddH2O 8.5 µl
10.0 µl
3. Add 15 ul of Mix A to each sample and run program “CHIPCHIP”:
Step1: 55°C 4 minutes
Step2: 72°C 3 minutes
Step3: 95°C 2 minutes
Step4: 95°C 30 seconds
Step5: 60°C 30 seconds
Step6: 72°C 1 minute
Step7: GOTO Step4 24 times
Step8: 72°C 5 minutes
Step9: 4°C HOLD
4. Midway through Step1, add 10 µl Mix B to each tube to hot start reactions. If necessary, pause program in Step1 so tubes remain at 55°C while adding Mix B.
5. After PCR is completed, make precipitation mix:
Precipitation Mix
Final Conc. Stock 1x Mix
750 mM 7.5 M Ammonium acetate 25.0 µl
90% Ethanol 225.0 µl
250.0 µl
6. Pool samples where appropriate. Add 250 µl precipitation mix per 50 µl of PCR reaction. Incubate 30 min at -80°C.
7. Spin at 20,000 x g for 10 minutes at 4°C to pellet DNA. Wash pellets with 500 µl of 80% EtOH.
8. Dry pellets and resuspend each in 50 µl H2O.
9. Measure DNA concentration with NanoDrop (use 10-fold dilutions, if necessary) and normalize all samples to 500 ng/µl.
VIIIb. Ligation-mediated PCR (medium-scale)
1. Mix 25 µl of IP sample and 25 µl WCE sample in PCR tubes.
2. Make two buffer mixes per rxn:
Mix A
Final Conc. Stock 1x Mix
1x 10X Thermopol buffer (NEB) 4.00 µl
250 nM dNTP mix (2.5 mM each) 5.00 µl
1 µM oligo oJW102 (40 µM) 1.25 µl
ddH2O 4.75 µl
15.00 µl
Mix B
Final Conc. Stock 1x Mix
1x 10X Thermopol buffer (NEB) 1.0 µl
2.5 U Taq polymerase (5U/µl) 0.5 µl
ddH2O 8.5 µl
10.0 µl
3. Add 15 ul of Mix A to each sample and run program “LMPCR1”:
Step1: 55°C 4 minutes
Step2: 72°C 3 minutes
Step3: 95°C 2 minutes
Step4: 95°C 30 seconds
Step5: 60°C 30 seconds
Step6: 72°C 1 minute
Step7: GOTO Step4 14 times
Step8: 72°C 5 minutes
Step9: 4°C HOLD
4. Midway through Step1, add 10 µl Mix B to each tube to hot start reactions. If necessary, pause program in Step1 so tubes remain at 55°C while adding Mix B.
5. Transfer product to 1.5 ml centrifuge tube and add 475 ul ddH20 (total ~525ul).
Partial expansion
The following steps are for medium-scale amplification (16 to 48 reactions) of IP and WCE DNA using template generated from the 15 round PCR described above.
10. Make up PCR mixes:
Final Conc. Stock 1x Mix
1x 10x Thermopol buffer (NEB) 5.00 µl
250 nM dNTP mix (2.5 mM each) 5.00 µl
1 µM oligo oJW102 (40 µM) 1.25 µl
2.5 U Taq polymerase (5U/µl) 0.25 µl
ddH2O 33.50 µl
template DNA 5.00 µl
50.00 µl
11. Aliquot 50 µl of PCR mix to individual PCR tubes.
12. Run program “LMPCR2”:
Step1: 95°C 2 minutes
Step2: 95°C 30 seconds
Step5: 60°C 30 seconds
Step6: 72°C 1 minute
Step7: GOTO Step2 24 times
Step8: 72°C 5 minutes
Step9: 4°C HOLD
13. Make precipitation mix:
Precipitation Mix
Final Conc. Stock 1x Mix
750 mM 7.5 M Ammonium acetate 25.0 µl
90% Ethanol 225.0 µl
250.0 µl
14. Pool samples where appropriate. Add 250 µl precipitation mix per 50 µl of PCR reaction. Incubate 30 min at -80°C.
15. Spin at 20,000 x g for 10 minutes at 4°C to pellet DNA. Wash pellets with 500 µl of 80% EtOH.
16. Dry pellets and resuspend each in 50 µl H2O.
17. Measure DNA concentration with NanoDrop (use 10-fold dilutions, if necessary) and normalize all samples to 500 ng/µl.
VIIIc. Ligation-mediated PCR (Large-scale)
1. Mix 25 µl of IP sample and 25 µl WCE sample in PCR tubes.
2. Make two buffer mixes per rxn:
Mix A
Final Conc. Stock 1x Mix
1x 10X Thermopol buffer (NEB) 4.00 µl
250 nM dNTP mix (2.5 mM each) 5.00 µl
1 µM oligo oJW102 (40 µM) 1.25 µl
ddH2O 4.75 µl
15.00 µl
Mix B
Final Conc. Stock 1x Mix
1x 10X Thermopol buffer (NEB) 1.0 µl
2.5 U Taq polymerase (5U/µl) 0.5 µl
ddH2O 8.5 µl
10.0 µl
3. Add 15 ul of Mix A to each sample and run program “LMPCR1”:
Step1: 55°C 4 minutes
Step2: 72°C 3 minutes
Step3: 95°C 2 minutes
Step4: 95°C 30 seconds
Step5: 60°C 30 seconds
Step6: 72°C 1 minute
Step7: GOTO Step4 14 times
Step8: 72°C 5 minutes
Step9: 4°C HOLD
4. Midway through Step1, add 10 µl Mix B to each tube to hot start reactions. If necessary, pause program in Step1 so tubes remain at 55°C while adding Mix B.
5. Transfer product to 1.5 ml centrifuge tube and add 475 ul ddH20 (total ~525ul).
Full expansion
The following steps are for large-scale amplification (96 reactions each) of IP and WCE DNA using template generated from the 15 round PCR described above.
6. Make up PCR mixes:
Final Conc. Stock 1x Mix For 2x96-well plate
1x 10x Thermopol buffer (NEB) 5.00 µl 1.00 ml
250 nM dNTP mix (2.5 mM each) 5.00 µl 1.00 ml
1 µM oligo oJW102 (40 µM) 1.25 µl 250.00 µl
2.5 U Taq polymerase (5U/µl) 0.25 µl 50.00 µl
ddH2O 33.50 µl 6.75 ml
45.00 µl 9.00 ml
7. Split PCR mix into two 4.5 ml aliquots. Add 500 µl of IP DNA to one and 500 µl of WCE DNA to the other.
8. Aliquot 50 µl of total mix per well for an entire 96-well plate. Use one plate for IP DNA and one plate for WCE DNA.
9. Run program “LMPCR2”:
Step1: 95°C 2 minutes
Step2: 95°C 30 seconds
Step5: 60°C 30 seconds
Step6: 72°C 1 minute
Step7: GOTO Step2 24 times
Step8: 72°C 5 minutes
Step9: 4°C HOLD
9. Pool all wells for IP DNA in a 50 ml conical tube. Add 2.5 ml of 7.5M ammonium acetate and 25 ml of EtOH. Repeat for WCE DNA.
10. Split each precipitate mix into two 30 ml Corex tubes. Cover tubes with parafilm. Incubate for 60 min at -80°C.
11. Place tubes in centrifuge (Sorval SS34 rotor). Use appropriate adaptors. Spin at 12,000 x g for 60 minutes at 4°C.
12. Decant supernatant. Add 1 ml of EtOH and dislodge pellet. If necessary, scrape side of tube and vortex vigorously.
13. Transfer pellet and EtOH to a 1.5 ml microfuge tube and spin at 20,000 x g for 5 minutes at 4°C to pellet DNA.
14. Remove supernatant and add 80% EtOH. Resuspend pellet and then spin at 20,000 x g for 5 minutes at 4°C to pellet DNA.
15. Dry pellets and resuspend each in 500 µl H2O.
16. Measure DNA concentration with NanoDrop (use 10-fold dilutions, if necessary) and normalize all samples to 500 ng/µl.
IX. Cy3/Cy5 labeling of IP/WCE material
This is a random-primed, Klenow-based extension protocol derived from Invitrogen’s CGH kit. Our protocol varies from the instructions provided by Invitrogen in both reaction volume and reagent concentrations. We perform 20 reactions per “30 reaction” Invitrogen kit. A pair of reactions (one for each dye) yields enough material for 1-2 hybridizations. To scale up for more arrays, we increase number—not volume—of individual reactions. For whole genome arrays, we performed reactions in 96-well PCR plates (requires 10 CGH kits and 5 tubes of each Cy dye).
1. Open requisite number of CGH kits and consolidate 2.5x Random Primer Solution, lowT dNTP mix, Klenow, and Stop Buffer.
2. To label both IP and WCE, make priming solution mix for each:
Final Conc. Stock 1x Mix 100x Mix
2 µg LM-PCR product (500 ng/ul) 4.0 µl 400.0 µl
1x 2.5x random primer solution 35.0 µl 3.5 ml
ddH2O 36.0 µl 3.6 ml
75.0 µl 7.5 ml
3. Vortex mixture for 30 seconds.
4. Aliquot 75 µl mix/well.
5. Place plates in thermal cycler preheated to 95°C. After 5 minutes at 95°C, immediately transfer plate to ice-water bath for 5 minutes to flash cool reagents.
6. While reactions are cooling, make up label mix:
(we typically use Cy5 mix with IP DNA and Cy3 with WCE DNA)
Final Conc. Stock 1x Mix 100x Mix
110/55 nM lowT dNTP Mix (1.2/0.6 mM) 8.2 µl 820 µl
17 µM Cy5- or Cy3-dUTP (1 mM) 1.5 µl 150 µl
60 U Klenow (40 U/ul) 1.5 µl 150 µl
ddH2O 1.8 µl 180 µl
13.0 ul 1300 µl
7. Vortex mixture for 30 seconds.
8. Aliquot 13 µl label mix/well. Pipette up and down multiple times to mix reagents.
9. Incubate for 3 hours at 37°C. Keep samples in dark.
10. Add 9 µl stop buffer to each well and mix.
At this point, many different clean-up methods can be used to separate dye and nucleotides from longer DNA fragments. For small-scale cleanup, we use bind and elute columns, either CGH columns (Invitrogen) or PCR cleanup columns (Qiagen) and follow manufacturer’s directions. For large-scale preparations, we have used the following protocol.
11. Transfer labeling reactions to Microcon YM-30 filter plate.
12. Spin at 3,000 x g for 15 minutes at room temperature. Filtration will take longer if centrifuge is cold.
13. Make sure DNA retentate is dry. Resuspend DNA in 60 µl ddH2O by pipetting up and down multiple times. Pool identical wells.
14. Measure DNA concentration and dye incorporation using NanoDrop (each plate should yield ~1mg DNA).
15. Labeled DNA can be frozen at this point. For long-term storage, precipitate DNA and leave in pellet form at -20°C shielded from light.
X. Array Hybridization
Note: Keep samples in dark as much as possible (use foil as necessary)
1. Thaw labeled DNA.
2. Combine IP and WCE DNA and add ddH2O to 120 µl total volume per hybridization. Each hybridization requires 5 – 10 µg each of labeled IP and WCE DNA. Maximize amount of DNA within volume limitations. If labeled DNA solutions are too dilute, the maximum volume acceptable at this point is 166.5 µl. Adjust water in Step 6 accordingly.
3. Make up master mix of control nucleic acids. Note that we use the control oligos at 0.1x and not 1x concentration as suggested by the manufacturer:
Final Conc. Stock 1x Mix
750 ng herring sperm DNA (100 ng/µl) 7.5 µl
40 µg yeast tRNA (8 µg/µl) 5.0 µl
10 µg Human Cot-1 DNA (1 µg/µl) 10.0 µl
0.1x Agilent control oligos (10x) 5.0 µl
27.5 µl
4. Add control mixture to sample and mix thoroughly.
5. Aliquot 147.5 ul of sample/control mix to individual Eppendorf tubes (one tube per array to be hybridized).
6. Make up buffer master mix. Add reagents in the order listed:
Final Conc. Stock 1x Mix
ddH2O 46.5 µl
50 mM Na-MES pH 6.9 (500 mM) 50.0 µl
500 mM NaCl (5 M) 50.0 µl
6 mM EDTA (500 mM) 6.0 µl
0.5% Sarcosine (5% -- ultrapure) 50.0 µl
30 % Formamide (ultrapure) 150.0 µl
352.5 µl
7. Add 352.5 ul room temperature buffer mix to each tube. Samples can be left at room temperature during this step.
8. Heat samples for 3 minutes at 95°C.
9. Transfer tubes to 40°C heat block and incubate for 15 minutes.
10. Spin tubes at 13,000 x g for 45 seconds at room temperature.
11. Assemble arrays as described by manufacturer (Agilent Technologies). Briefly, slides bearing gaskets are placed face-up in a hybridization chamber. Hybridization solution is added to the slide and arrays are then placed face-down on the solution. The chamber is assembled and checked for free rotation of hybridization solution.
12. Incubate at 40°C in rotating oven for 20 hours.
XI. Array Washing
Note: Process one array at a time through Step 3. Steps 1 through 3 should be done quickly. Volumes and times here are for sets of 10 arrays during Steps 4 through 9 and may require optimization if you wish to process more or fewer slides.
1. Disassemble hybridization chambers and transfer sandwiched slides to reservoir containing 1 liter of Wash Buffer I.
2. While submerged, IMMEDIATELY and gently separate gasket slide from array slide. Wash array slide with 2 seconds of gentle agitation.
3. Place array in a metal, 30-slide rack submerged in a second dish of 850 ml of Wash I. Separate slides by 2-4 slots in the rack to allow for proper washing. This high volume will result in some spillage in Step 4, but is required to keep the slide submerged as you place it in the rack.
4. Wash for 5 minutes by placing dish on orbital shaker set at 60 rpm.
5. During the wash, set up two dishes, one containing 650 ml of Wash II and one containing 750 ml of Wash II. The second dish will be placed on a magnetic stir plate. Check that a stir bar will be able to move freely when the rack is placed inside.
6. After Step 4, smoothly and rapidly remove slide rack, dip for 1 second in the dish containing 650 ml of Wash II and transfer to the second dish containing 750 ml of Wash II and a stir bar.
7. Wash for 5 minutes with stirring (typically setting 4 on most stir plates).
8. During the wash, set up two dishes, one containing 650 ml of 100% acetonitrile and one containing 750 of Agilent Wash III (follow manufacturer’s directions for preparation of Wash III).
9. After Step 7, remove rack, dip for 1 second in dish containing acetonitrile and transfer to Wash III. Wash for 30 seconds with stirring (setting 4 on most stir plates).
10. SLOWLY and evenly remove rack and slides from Wash III. It should take approximately 10 seconds to lift rack from solution (the slides should be dry at this point).
11. Scan immediately or store in sealed high N2 environment.
Wash I
Final Conc. Stock For 2000 ml
6x 20x SSPE 600.0 ml
ddH2O 1398.0 ml
0.005% 5% N-lauroylsarcosine (ultrapure) 2.0 ml
2000.0 ml
Wash II
Final Conc. Stock For 2000 ml
0.06x 20x SSPE 6.0 ml
ddH2O 1994.0 ml
2000.0 ml
Wash III
(Purchase 1x solution from Agilent)
Appendix:
Oligos for blunt end ligation:
oJW102: 5’-GCGGTGACCCGGGAGATCTGAATTC
oJW103: 5’-GAATTCAGATC
Preparation of 15 µM Linker Stock:
1. Mix the following:
250 µl Tris-HCl (1M) pH 7.9
375 µl oligo oJW102 (40 µM stock)
375 µl oligo oJW103 (40 µM stock)
NOTE: Order these oligos desiccated, then resuspend in ddH20 to 40 µM.
2. Make 50 or 100 ul aliquots in PCR tubes.
3. Place in a thermal cycler and run program LINKER:
95°C for 5 minutes
70°C for 1 minute
Ramp down to 4°C (0.4°C/min)
4°C HOLD
4. Store linkers at -20 °C.
