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CHIP Reminder Wear Gloves, lab coat and eye protection!!! Day 1 1. Stimulate or treat at least 1 x 106 cells on a 10cm dish. For LIF, treat at 50ng/mL to 100ng/mL for 20 minutes as 37ºC. 2. Crosslink histones by adding formaldehyde directly to culture medium for a final concentration of 1%. For 10cm plate with 10mL media, add 270uL of 37% formaldehyde. 3. Incubate at room temperature for 20 minutes. 4. Take off 7mL of media (leaving 3mL on the plate). Scrape cells in the 3mL and transfer to a 15mL conical. 5. Pellet cells for 4 minutes at 2000 rpm at 4ºC. 6. Aspirate off media, add 5mL of ice cold 1x PBS. Resupend by vortexing. Pellet for 4 minutes at 2000 rpm at 4ºC. 7. Repeat 2 times. 8. Resuspend pellet in 150uL of SDS lysis buffer supplemented with protease and phosphatase inhibitors (PMSF, aprotinin, leupeptin, pepstatin, DTT, NaF, Na3VO4). Transfer lysate to eppendorf. 9. Incubate on ice for 10 minutes. 10. Sonication - Tune sonicator before use. - Keep tip almost at bottom but not touching tube. - Keep on ice at all times make a mound around tube to hold it in place. - Sonicate 3 times on control setting 3 for 20 seconds each. 11. Centrifuge samples for 10 minutes at 13,000 rpm and add 150uL of the sonicated cell supernatant to a new 1.5mL microcentrifuge tube. Discard pellet. 12. Dilute the sonicated cell supernatant by adding 1350uL CHIP dilution buffer supplemented with aforementioned protease and phosphatase inhibitor (see step 8). 13. Remove 15uL of the diluted supernatant to keep as your input fraction. Keep at 4ºC until ready to reverse crosslinks. 14. Pre-clear the 1.5mL of the diluted supernatant with 60uL of salmon sperm DNA/protein Agarose 50% slurry for 30 minutes at 4ºC with rotation (use pipet tip with end that has been cut off). 15. Pellet agarose by spinning for 1 minute at 1000 rpm and then collect the supernatant fraction. 16. Add the immunoprecipitating Ab. Incubate overnight at 4ºC with rotation. Day 2 17. Add 45uL of the slurry and collect the Ab/histone complex by rotating at 4ºC for one hour. 18. Pellet agarose for 1 minute at 1000 rpm at 4ºC. Carefully aspirate off non-specific supernatant. 19. Wash by rotating in homemade eppendorf tube rotator for 3-5 minutes with each of the following buffers, spinning down after each with a 1 minute, 1000 rpm spin at 4¡C. a. Lo Salt wash (2 times) b. Hi Salt wash (2 times) c. LiCl wash (2 times) d. 1 x TE (3 times) Note: Keep buffers at 4ºC. 20. Freshly prepare elution buffer (1% SDS and 0.1M NaHCO3). 21. Elute by adding 200uL of elution buffer to the pelleted complex. Vortex briefly and mix occasionally for 15 minutes at room temperature. 22. Spin down agarose at room temperature (1 minute), 1000 rpm) and transfer the supernatant to a new collection tube. Repeat elution to pelleted complex. Combine the 2 eluates. 23. Add 16uL of 5M NaCl to the combined eluate (400uL), and 0.6uL 5M NaCl to the saved input fractions. 24. Reverse histone crosslinks by heating at 65ºC for 4 hours. (After this step, sample can be stored at -20ºC. 25. To the sample add 8uL of 0.5M EDTA, 16uL of 1M Tris-CL and 1.6uL of 10mg/mL Proteinase K. To the input add 0.3uL of 0.5M EDTA, 0.6uL of 1M Tris-HCL pH 6.5 and 0.6uL of a 10x dilution of 10mg/mL proteinase K. 26. Incubate for 1 hour at 45ºC. 27. Bring up volume of inputs with TE. 28. Extract DNA once with 1 volume of phenol/chloroform. Extract 2 times with ½ volume straight chloroform. 29. Add 10% 3M NaOAc. Then add 2 volumes of 100% EtOH. Add 1uL of stock glycogen. 30. Mix vigorously and place at -80ºC for at least 1 hour. 31. Spin down at max speed for 20 minutes. 32. Wash pellet with 70% EtOH. 33. Resuspend in 10uL of TE. 34. Proceed to PCR. CHIP Solutions SDS Lysis Buffer 1mL 10% SDS 200uL 0.5M EDTA 500uL 1M Tris HCl pH 8.0 8.3mL ddH20 Total Volume: 10mL CHIP Dilution Buffer 50uL 10% SDS 0.55mL Triton-X 100 120uL 0.5M EDTA 835uL 1M Tris HCl pH 8.0 1.67mL 5M NaCl 46.8mL ddH20 Total Volume: 50mL Lo Salt 0.5mL 10% SDS 0.5mL Triton-X 100 200uL 0.5 M EDTA 1mL 1M Tris-HCl pH 8.0 1.5mL 5M NaCl 46.3mL ddH20 Total Volume 50mL Hi Salt 0.5mL 10% SDS 0.5mL Triton-X 100 200uL o.5M EDTA 1mL 1M Tris-HCl pH 8.0 5mL 5M NaCl 42.8mL ddH20 Total Volume: 50mL LiCl 2.5mL 5M LiCl 0.5mL NP-40 0.5g Deoxychloric Acid 100uL 0.5M EDTA 0.5mL Tris-HCl 46.4mL ddH20 Total Volume: 50mL 1x TE pH8 1mL Tris-HCl 0.2mL 0.5M EDTA 98.8mL ddH20 Total Volume: 100mL |
