Changes

* [[Dilution Buffer]]*[[Low Salt Immune Complex Wash Buffer]] * [[High Salt Immune Complex Wash Buffer]] * [[LiCl Immune Complex Wash Buffer]] make fresh (cold)* [[TE: 10 mM Tris 7.5, 0.1 mM EDTA (cold)Buffer]]

* If using 10cm dishes add 270ul 250ul of 3740% formaldehyde

(60~45-50% amplitude), with at least 30 second cooling on ice between each 30-second sanitation. Remember to clean sonicator with water prior to use, in between samples and following use.

# Add 20 μl re-suspended magnetic bead slurry (for each sample you plan to use with this antibody) to a 115.5 ml microfuge tube on ice Prepare enough Dilution Buffer containing 1 ml cold PBS/BSA. Vortex briefly to mix well.# Place protease inhibitors for the microfuge tubes on the magnet rack number of desired immunoprecipitations and remove supernatants.# Resuspend the beads in 1 ml cold PBS/BSA.# Repeat Steps b and c 3 times.# Add 200 μl PBS/BSA (for each sample you plan to use this antibody with) to beads.# Add 1 μg primary antibody. Mix gently by tapping--Do not vortex beads after adding the antibody.# Gently mix store on a rotator platform for at least 2 hours at 4°C.# Wash beads 3 times (steps b-c), resuspending the beads by inverting the tubes during each wash.# Resuspend in 100 μl PBS/BSA (for each sample you plan to use this antibody with), and proceed to Step 2ice.

==== Incubate bead-antibody complex with fragmented, cross-linked chromatin====# Add 100 μl of antibody-coupled beads (from step 116.i above) to each 25ug chromatin Each IP requires the addition of preparation (from Sonication protocol) 900 μL of Dilution Buffer and incubate on a rotator for '''one hour at room temperature''', followed by '''one hour at 4°C'''4.# Collect beads containing immuno-bound chromatin by placing the microfuge tube on a magnet rack.# Remove and discard supernatant.# Wash beads 5 times with 1 mL LiCl Wash Buffer, mixing 3 minutes for each wash on a rotator.# Add 1 ml TE Buffer to beads. Mix 1 minute on rotator and then place tubes on magnet rack to collect beads and discard supernatant.# Resuspend the bead pellet in 200 μl IP Elution Buffer (at room temperature). Vortex to mixμL of Protease Inhibitor Cocktail II.

==== Reverse cross17. Immunoprecipitations should include the positive control (Anti-linking RNA Polymerase II), and recover ChIP DNA ====the negative control, (Normal Mouse IgG), and the antibody of interest (user supplied). It is recommended that the user include a negative control IgG of the same species as the antibody of interest.# Incubate bead pellet from * Prepare one microfuge tube containing 100 μL of sheared crosslinked chromatin (Section B, step 25) for the number of desired immunoprecipitations and put on ice.f above If chromatin has been previously frozen, thaw on ice.* Alternatively, if multiple immunoprecipitations will be performed from the same chromatin preparation, place the entire volume for the number of desired immunoprecipitations in one large tube that will be able to accommodate a 65°C water bath volume of 1.1 mL for each IP.* Each 100 μL will contain ~1 hourx 106 cell equivalents of chromatin. 18. Add 900 μL of Dilution Buffer containing Protease Inhibitor Cocktail II into each tube containing 100 μL of chromatin.* Alternatively, shake or vortex every 15 minutes if multiple immunoprecipitations will be performed from the same chromatin preparation, use the appropriate volume of Dilution Buffer containing Protease Inhibitor Cocktail II for the correct number of immunoprecipitations. 19. Add 60 μL of Protein G Agarose for each IP.* The Protein G Agarose is a 50% slurry. Gently mix by inversion before pipetting.* This step serves to elute “preclear” the immuno-bound chromatin , i.e., to remove proteins or DNA that may bind nonspecifically to the Protein G agarose.* Alternatively, if multiple immunoprecipitations will be performed from the same chromatin preparation, use the appropriate volume of Protein G Agarose for the correct number of immunoprecipitations. 20. Incubate for '''1 hour''' at 4°C with rotation. 21. Pellet agarose by brief centrifugation (3000-5000 x g for 1 minute). * Do not spin Protein G Agarose beadsat high speeds. Applying excessive g-force may crush or deform the beads and cause them to pellet inconsistently.# Spin 22. Remove 10 μL (1%) of the supernatant as Input and save at 144°C until Section D,000 rpm in a microfuge at room temperature for 3 minutesstep 1.# * If different chromatin preparations are being carried together through this protocol, remove1% of the chromatin as Input from each. 23. Collect the remaining supernatantand dispense 1 mL aliquots into fresh microfuge tubes. Discard agarose pellet. 24. Add the immunoprecipitating antibody to the supernatant fraction:* For the positive control, which contains anti-RNA Polymerase, add 1.0 μg of antibody per tube.* For the ChIP’d DNAnegative control, Normal Mouse IgG, add 1.0 μg of antibody per tube.* For user-provided antibody and controls, add between 1-10 μg of antibody per tube. The tubes can appropriate amount of antibody needs to be placed on determined empirically. 25. Incubate '''overnight''' at 4°C with rotation.* It may be possible to reduce the magnet incubation time of the IP. This depends on many factors(antibody, gene target, cell type, etc.) and will have to facilitate supernatant recoverybe tested empirically.# Incubate 26. Add 60 μL of Protein G Agarose to each IP and incubate for '''1 hour''' at 4°C with rotation.* This serves to collect the antibody/antigen/DNA complex. 27. Pellet Protein G Agarose by brief centrifugation (3000-5000 x g for 1 minute) and remove thesupernatant containing fraction. 28. Wash the ChIP’d DNA Protein G Agarose-antibody/chromatin complex by resuspending the beads in 1 mL each of the cold buffers in the order listed below and incubating for '''3-5 minutes''' on a 65°C rotating platform followed by brief centrifugation (3000-5000 x g for 1 minute) and careful removal of the supernatant fraction:** [[Low Salt Immune Complex Wash Buffer]] (Catalog # 20-154), '''one wash'''** [[High Salt Immune Complex Wash Buffer]] (Catalog # 20-155), '''one wash'''** [[LiCl Immune Complex Wash Buffer]] (Catalog # 20-156), '''3-5 washes'''** [[TE Buffer]] (Catalog # 20-157), '''two washes''' ''Note: for TE washes use a pipette to carefully aspirate, the beads seem to come off of the magnet easily with this wash'' === Elution of Protein/DNA Complexes ======== Prior to starting this section: =====* Bring 1 M NaHCO3 to room temperature. A precipitate may be observed but will go into solution once room temperature is achieved. The 1 M NaHCO3 can be vortexed.* Set water bath overnight to complete 65°C for use later.29. Make Elution Buffer for all IP tubes as well as all Input tubes.* For each tube, prepare 200 μL of elution buffer as follows: 10 μL 20% SDS, 20 μL 1 M NaHCO3 and 170 μL sterile, distilled water.* OR can make this way [[ChIP Elution Buffer]]* Alternatively, make a large volume to accommodate all tubes. For example, if there are 10 tubes mix together 105 μL 20% SDS, 210 μL 1M NaHCO3 and 1.785 mL sterile, distilled water. 30. For Input tubes, add 200 μL of Elution Buffer and set aside at room temperature. 31. Add 100 μL of Elution Buffer to each tube containing the reversal antibody/agarose complex. Mix by flicking tube gently. 32. Incubate at room temperature for '''15 minutes'''. 33. Pellet agarose by brief centrifugation (3000-5000 x g for 1 minute) and collect supernatant into new microfuge tubes. 34. Repeat steps 4-6 and combine eluates (total volume = 200 μL). === Reverse Crosslinks of Protein/DNA Complexes to Free DNA===36. To all tubes (IPs and Inputs) add 8 μL 5 M NaCl and incubate at 65°C for 4-5 hours or '''overnight''' to reverse the formaldehyde crossDNA – Protein crosslinks. After this step the sample can be stored at -links20°C and the protocol continued the next day. 37. To all tubes, add 1 μL of RNase A and incubate for '''30 minutes''' at 37°C. 38. Add 4 μL 0.5M EDTA, 8 μL 1M Tris-HCl and 1 μL Proteinase K to each tube and incubate at 45°C for'''1-2 hours'''.

# 39. Add 5 volumes Qiagen Buffer PB (QIAquick PCR Purification Kit) to one volume of ChIP’d DNA. Add pH detector (at a 1:250 dilution) to samples. Upon addition of Buffer PB, the sample should be yellow, indicating the correct pH. If the sample is not yellow, the pH should be adjusted with 3M sodium acetate as recommended by the manufacturer (Qiagen). One microliter at a time, mixing between each works fine.# 40. Add half (~600 µl) of the solution to a QIAquick PCR Purification column, centrifuge for 30-60 sec @ 13,000 RPM , and then repeat with other half to bind the ~1.2 ml sample on a Qiagen column.# 41. Wash the column with 750 µl Qiagen Buffer PE, centrifuge for 30-60sec @ 13,000 RPM, empty, . 42. Empty the collection tube and centrifuge the column containing the bound DNA a second time to allow it to dry.# 43. Elute the DNA from the column with two 35 µl aliquots (note: this is how much you will need to run duplicates with 5 primers and may need to be adjusted based on your experiment) of warmed (~55°C) Qiagen Buffer EB, allow to sit on column for 1 minute, spin for 1 min @ 13,000 RPM, and repeat).