染色質免疫共沉淀 ChIP Protocol及crosslink 的原理.doc

ChIP ProtocolI. Formaldehyde cross-linking of cellsUse 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 4C (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 4C (Sorvall Legend RT centrifuge with swinging bucket rotor). Discard supernatant.7.Flash freeze cells in liquid nitrogen and store pellets at 80C.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 4C.5.Pool cells in required number of 50 ml conical tubes and spin at 1,350 x g for 5 minutes at 4C (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 4C. 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 4C (Sorvall Legend RT centrifuge with swinging bucket rotor). Discard supernatant.8.Flash freeze cells in liquid nitrogen and store pellets at 80C.Formaldehyde SolutionFinal Conc.StockFor 50 ml50 mM1M Hepes-KOH, pH 7.52.5 ml100 mM5M NaCl1.0 ml1 mM0.5M EDTA50.0 l0.5 mM0.5M EGTA100.0 l11%37% Formaldehyde14.9 mlddH2O31.5 mlII. Preblock and binding of antibody to magnetic beadsNOTE: 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 l 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 l block solution and add 10 g of antibody.5. Incubate overnight on a rotating platform at 4C.6. Next day, wash beads as described above (3 times in 1 ml block solution).7. Resuspend in 100 l block solution.Block SolutionFinal Conc.StockFor 100 ml1x10x PBS10.0 ml0.5% BSA (w/v)BSA500.0 mgddH2O90.0 ml100.0 mlIII. Cell Sonication 1. Resuspend each pellet of 108 cells in 5 ml of LB1. Rock at 4C for 10 min. Spin at 1,350 x g for 5 minutes at 4C 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 4C.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 l 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 4C 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 l of cell lysate from each sample as WCE DNA. Store at -20C.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 -20C.)Lysis Buffer 1 (LB1)Final Conc.StockFor 100 ml50 mM1M Hepes-KOH, pH 7.55.0 ml140 mM5M NaCl2.8 ml1 mM0.5M EDTA0.2 ml10%50% glycerol20.0 ml0.5%10% NP-405.0 ml0.25%10% Triton X-1002.5 mlddH2O64.5 mlLysis Buffer 2 (LB2)Final Conc.StockFor 100 ml10 mMTris-HCl, pH 8.01.0 ml200 mM5M NaCl4.0 ml1 mM0.5M EDTA0.2 ml0.5 mM0.5M EGTA0.1 mlddH2O94.7 mlLysis Buffer 3 (LB3)Final Conc.StockFor 100 ml10 mMTris-HCl, pH 8.01.0 ml100 mM5M NaCl2.0 ml1 mM0.5M EDTA0.2 ml0.5 mM0.5M EGTA0.1 ml0.1%10% Na-Deoxycholate1.0 ml0.5%20% N-lauroylsarcosine2.5 mlddH2O93.2 mlIV. Chromatin immunoprecipitation1.Add 100 l 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 4C.V. Wash, elution, and cross-link reversalSteps 1 through 6 should be done in a 4C 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 beads4. 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 4C and remove any residual TE buffer.7. Add 210 l of elution buffer.8. Elute at 65C 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 l of supernatant and transfer to new tube. Reverse crosslink（交聯(lián)逆轉，即將交聯(lián)打開） of this IP DNA by incubating at 65C overnight.11. Thaw 50 l of WCE reserved after sonication, add 150 l (3 volumes) of elution buffer and mix. Reverse crosslink of this WCE DNA by incubating at 65C overnight.Wash Buffer (RIPA)Final Conc.StockFor 250 ml50 mM1M Hepes-KOH, pH 7.612.5 ml500 mM5M LiCl25.0 ml1 mM0.5M EDTA0.5 ml1%10% NP-4025.0 ml0.7%10% Na-Deoxycholate17.5 mlddH2O169.5 mlElution BufferFinal Conc.StockFor 100 ml50 mM1M Tris-HCl, pH 8.05.0 ml10 mM0.5M EDTA2.0 ml1%10% SDS10.0 mlddH2O83.0 mlVI. Digestion of cellular protein and RNA（得到純DNA）1.Add 200 l of TE to each tube of IP and WCE DNA to dilute SDS in elution buffer.2.Add 8 l of 10 mg/ml RNaseA (0.2 g/ml final concentration).3.Mix and incubate at 37C for 2 hours.4.Add 4 l of 20 mg/ml proteinaseK (0.2 g/ml final concentration).5.Mix and incubate at 55C 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 l of 5M NaCl (200 mM final concentration) and 1.5 l of 20 g/l glycogen (30 g total).8. Add 800 l EtOH. Incubate for 30 min at -80C.9. Spin at 20,000 x g for 10 minutes at 4C to pellet DNA. Wash pellets with 500 l of 80% EtOH.10. Dry pellets and resuspend each in 70 l of 10mM Tris-HCl, pH 8.0.11. Save 15 l 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/ulVII. T4 Fill-in and blunt-end ligationA. T4 polymerase blunting of DNA ends Steps 1 through 6 should be kept on ice.1. Mix 2 l (200 ng) WCE DNA and 53 l ddH2O for each IP.2. Aliquot 55 l for each IP sample. Keep all tubes on ice.3. Make blunting master mix on ice:Final Conc.Stock1x Mix1x10x T4 DNA polymerase buffer11.0 l5 ug10 g/l BSA (NEB)0.5 ul40 nM10mM each dNTP1.0 l1.5 U3U/l T4 DNA polymerase (NEB)0.5 lddH2O42.0 l55.0 l4. Add 55 l of blunting master mix to all samples.5. Incubate for 20 minutes at 12C in thermal cycler.6. Add 11.5 l of 3M sodium acetate, pH 8.0 and 0.5 l of 20 g/l glycogen (10 g total).7. Transfer reaction to pre-chilled Phaselock tubes and extract 1x with 120 l P:C:IA (follow instructions provided by Eppendorf).8. Transfer aqueous layer to new centrifuge tube containing 250 l EtOH. Incubate for 30 minutes at -80C.9. Spin at 20,000 x g for 10 minutes at 4C to pellet DNA. Wash pellets with 500 l of 80% EtOH.10. Dry pellets and resuspend each in 25 l H2O. Chill on ice.B. Blunt-end ligation1. Make ligase master mix on ice (25 l per reaction):Final Conc.Stock1x Mix1x5x ligase buffer10.0 l2 M15 M linkers (see Appendix)6.7 ul200U400U/l T4 DNA ligase (NEB)0.5 lddH2O7.8 l55.0 l2. Add 25 l mix to 25 l of sample.3. Incubate 16 hours in 16C water bath.4. Add 6 l of 3M sodium acetate and 130 l EtOH. Incubate 30 min at -80C.5. Spin at 20,000 x g for 10 minutes at 4C to pellet DNA. Wash pellets with 500 l 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 l of IP sample and 25 l WCE sample in PCR tubes.2. Make two buffer mixes per rxn:Mix AFinal Conc.Stock1x Mix1x10X Thermopol buffer (NEB)4.00 l250 nMdNTP mix (2.5 mM each)5.00 l1 Moligo oJW102 (40 M)1.25 lddH2O4.75 l15.00 lMix BFinal Conc.Stock1x Mix1x10X Thermopol buffer (NEB)1.0 l2.5 UTaq polymerase (5U/l)0.5 lddH2O8.5 l10.0 l3. Add 15 ul of Mix A to each sample and run program “CHIPCHIP”: Step1:55C4 minutesStep2:72C3 minutesStep3:95C2 minutesStep4:95C30 secondsStep5:60C30 secondsStep6:72C1 minuteStep7:GOTO Step4 24 timesStep8:72C5 minutesStep9:4CHOLD4. Midway through Step1, add 10 l Mix B to each tube to hot start reactions. If necessary, pause program in Step1 so tubes remain at 55C while adding Mix B.5. After PCR is completed, make precipitation mix:Precipitation MixFinal Conc.Stock1x Mix750 mM7.5 M Ammonium acetate25.0 l90%Ethanol225.0 l250.0 l6. Pool samples where appropriate. Add 250 l precipitation mix per 50 l of PCR reaction. Incubate 30 min at -80C.7. Spin at 20,000 x g for 10 minutes at 4C to pellet DNA. Wash pellets with 500 l of 80% EtOH.8. Dry pellets and resuspend each in 50 l H2O.9. Measure DNA concentration with NanoDrop (use 10-fold dilutions, if necessary) and normalize all samples to 500 ng/l. VIIIb. Ligation-mediated PCR (medium-scale)1.Mix 25 l of IP sample and 25 l WCE sample in PCR tubes.2.Make two buffer mixes per rxn:Mix AFinal Conc.Stock1x Mix1x10X Thermopol buffer (NEB)4.00 l250 nMdNTP mix (2.5 mM each)5.00 l1 Moligo oJW102 (40 M)1.25 lddH2O4.75 l15.00 lMix BFinal Conc.Stock1x Mix1x10X Thermopol buffer (NEB)1.0 l2.5 UTaq polymerase (5U/l)0.5 lddH2O8.5 l10.0 l3.Add 15 ul of Mix A to each sample and run program “LMPCR1”: Step1:55C4 minutesStep2:72C3 minutesStep3:95C2 minutesStep4:95C30 secondsStep5:60C30 secondsStep6:72C1 minuteStep7:GOTO Step4 14 timesStep8:72C5 minutesStep9:4CHOLD4.Midway through Step1, add 10 l Mix B to each tube to hot start reactions. If necessary, pause program in Step1 so tubes remain at 55C while adding Mix B.5.Transfer product to 1.5 ml centrifuge tube and add 475 ul ddH20 (total 525ul). Partial expansionThe 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.Stock1x Mix1x10x Thermopol buffer (NEB)5.00 l250 nMdNTP mix (2.5 mM each)5.00 l1 Moligo oJW102 (40 M)1.25 l2.5 UTaq polymerase (5U/l)0.25 lddH2O33.50 ltemplate DNA5.00 l50.00 l11. Aliquot 50 l of PCR mix to individual PCR tubes.12. Run program “LMPCR2”:Step1:95C2 minutesStep2:95C30 secondsStep5:60C30 secondsStep6:72C1 minuteStep7:GOTO Step2 24 timesStep8:72C5 minutesStep9:4CHOLD13. Make precipitation mix:Precipitation MixFinal Conc.Stock1x Mix750 mM7.5 M Ammonium acetate25.0 l90%Ethanol225.0 l250.0 l14. Pool samples where appropriate. Add 250 l precipitation mix per 50 l of PCR reaction. Incubate 30 min at -80C.15. Spin at 20,000 x g for 10 minutes at 4C to pellet DNA. Wash pellets with 500 l of 80% EtOH.16. Dry pellets and resuspend each in 50 l H2O.17. Measure DNA concentration with NanoDrop (use 10-fold dilutions, if necessary) and normalize all samples to 500 ng/l.VIIIc. Ligation-mediated PCR (Large-scale)1.Mix 25 l of IP sample and 25 l WCE sample in PCR tubes.2.Make two buffer mixes per rxn:Mix AFinal Conc.Stock1x Mix1x10X Thermopol buffer (NEB)4.00 l250 nMdNTP mix (2.5 mM each)5.00 l1 Moligo oJW102 (40 M)1.25 lddH2O4.75 l15.00 lMix BFinal Conc.Stock1x Mix1x10X Thermopol buffer (NEB)1.0 l2.5 UTaq polymerase (5U/l)0.5 lddH2O8.5 l10.0 l3.Add 15 ul of Mix A to each sample and run program “LMPCR1”: Step1:55C4 minutesStep2:72C3 minutesStep3:95C2 minutesStep4:95C30 secondsStep5:60C30 secondsStep6:72C1 minuteStep7:GOTO Step4 14 timesStep8:72C5 minutesStep9:4CHOLD4.Midway through Step1, add 10 l Mix B to each tube to hot start reactions. If necessary, pause program in Step1 so tubes remain at 55C while adding Mix B.5.Transfer product to 1.5 ml centrifuge tube and add 475 ul ddH20 (total 525ul). Full expansionThe 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.Stock1x MixFor 2x96-well plate1x10x Thermopol buffer (NEB)5.00 l1.00 ml250 nMdNTP mix (2.5 mM each)5.00 l1.00 ml1 Moligo oJW102 (40 M)1.25 l250.00 l2.5 UTaq polymerase (5U/l)0.25 l50.00 lddH2O33.50 l6.75 ml45.00 l9.00 ml7. Split PCR mix into two 4.5 ml aliquots. Add 500 l of IP DNA to one and 500 l of WCE DNA to the other.8. Aliquot 50 l 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”:Step1:95C2 minutesStep2:95C30 secondsStep5:60C30 secondsStep6:72C1 minuteStep7:GOTO Step2 24 timesStep8:72C5 minutesStep9:4CHOLD9. 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 -80C.11. Place tubes in centrifuge (Sorval SS34 rotor). Use appropriate adaptors. Spin at 12,000 x g for 60 minutes at 4C.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 4C to pellet DNA.14. Remove supernatant and add 80% EtOH. Resuspend pellet and then spin at 20,000 x g for 5 minutes at 4C to pellet DNA.15. Dry pellets and resuspend each in 500 l H2O.16. Measure DNA concentration with NanoDrop (use 10-fold dilutions, if necessary) and normalize all samples to 500 ng/l. IX. Cy3/Cy5 labeling of IP/WCE materialThis is a random-primed, Klenow-based extension protocol derived from Invitrogens 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 numbernot volumeof 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. Stock1x Mix100x Mix2 gLM-PCR product (500 ng/ul)4.0 l400.0 l1x2.5x random primer solution35.0 l3.5 mlddH2O36.0 l