Changes

# Add 20 μl re1. Prepare enough Dilution Buffer containing protease inhibitors for the number of desired immunoprecipitations and store on ice.• Each IP requires the addition of 900 μL of Dilution Buffer and 4.5 μL of Protease Inhibitor Cocktail II.• Immunoprecipitations should include the positive control (Anti-suspended magnetic bead slurry RNA Polymerase II), and the negative control, (for each sample you plan to use with this Normal Mouse IgG), and the antibodyof interest (user supplied) to . It is recommended that the user include a 1negative control IgG of the same species as the antibody of interest.5 ml 2. Prepare one microfuge tube containing 100 μL of sheared crosslinked chromatin (Section B, step 5) for the number of desired immunoprecipitations and put on ice containing 1 ml cold PBS/BSA. Mix wellIf chromatin has been previously frozen, '''do not vortex'''thaw on ice. ''Note: For each wash• Alternatively, you if multiple immunoprecipitations will want to invert be performed from the tube while on same chromatin preparation, place the magnet rack to collect entire volume for the beads that remain number of desired immunoprecipitations in one large tube that will be able to accommodate a volume of 1.1 mL for each IP.• Each 100 μL will contain ~1 x 106 cell equivalents of chromatin.3. Add 900 μL of Dilution Buffer containing Protease Inhibitor Cocktail II into each tube containing 100 μL of chromatin.• Alternatively, if multiple immunoprecipitations will be performed from the liquid at same chromatin preparation, use the top appropriate volume of Dilution Buffer containing Protease Inhibitor Cocktail II for the tubecorrect number of immunoprecipitations. 4. 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 will allow you aspirate step serves to “preclear” the liquid 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 top same chromatin preparation, use the appropriate volume of Protein G Agarose for the tube between washes without sucking up any correct number of immunoprecipitations.5. Incubate for 1 hour at 4°C with rotation.6. Pellet agarose by brief centrifugation (3000-5000 x g for 1 minute).• Do not spin Protein G Agarose beads at high speeds. Applying excessive g-force may crush or deform the beadsand cause them to pellet inconsistently.''# Place 7. Remove 10 μL (1%) of the microfuge tubes on the magnet rack supernatant as Input and remove supernatantssave at 4°C until Section D, step 1.# Resuspend the beads in • If different chromatin preparations are being carried together through this protocol, remove1 ml cold PBS/BSA% of the chromatin as Input from each.# Repeat Steps b 8. Collect the remaining supernatant and c 3 timesdispense 1 mL aliquots into fresh microfuge tubes. Discard agarose pellet.# 9. Add 200 μl PBS/BSA (for each sample you plan to use this the immunoprecipitating antibody with) to beadsthe supernatant fraction:• For the positive control, anti-RNA Polymerase, add 1.0 μg of antibody per tube.# Add • For the negative control, Normal Mouse IgG, add 1 .0 μg primary of antibodyper tube. Mix gently by tapping• For user-provided antibody and controls, add between 1-'''Do not vortex beads'''10 μg of antibody per tube. The appropriate amount of antibody needs to be determined empirically.# Gently mix on a rotator platform for at least '''2 hours''' 10. Incubate overnight at 4°Cwith rotation.# Wash beads 3 times (steps b-c), resuspending • It may be possible to reduce the beads by inverting incubation time of the tubes during each washIP.This depends on many factors# Resuspend in 100 μl PBS/BSA (for each sample you plan to use this antibody with, gene target, cell type, etc.), and proceed will have to Step 2be tested empirically. ==== Incubate bead-antibody complex with fragmented, cross-linked chromatin====# 11. Add 100 μl 60 μL of antibody-coupled beads from above Protein G Agarose to each 25ug chromatin of preparation (from Sonication protocol) IP and incubate on a rotator for '''1 hour at room temperature''', followed 4°C with rotation.• This serves to collect the antibody/antigen/DNA complex.12. Pellet Protein G Agarose by '''brief centrifugation (3000-5000 x g for 1 hour at 4°C'''minute) and remove thesupernatant fraction.# Collect beads containing immuno13. Wash the Protein G Agarose-bound antibody/chromatin complex by placing resuspending the microfuge tube beads in 1 mL each of the cold buffers in the order listed below and incubating for 3-5 minutes on a magnet rackrotating platform followed by brief centrifugation (3000-5000 x g for 1 minute) and careful removal of the supernatant fraction:a.Low Salt Immune Complex Wash Buffer (Catalog # 20-154), one washb. High Salt Immune Complex Wash Buffer (Catalog # Remove and discard supernatant20-155), one washc. ''Turn LiCl Immune Complex Wash Buffer (Catalog # 20-156), one washd. TE Buffer (Catalog # 20-157), two washesD. Elution of Protein/DNA ComplexesPrior 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 to 65°C before next step''for use in Section E.# Wash beads 5 times with 1 mL LiCl Wash . Make Elution Buffer, mixing 3 minutes for each wash on a rotatorall IP tubes as well as all Input tubes (see Section C, step 7).# Add • For each tube, prepare 200 μL of elution buffer as follows: 10 μL 20% SDS, 20 μL 1 ml TE Buffer M NaHCO3 and 170 μL sterile, distilled water.2. Alternatively, make a large volume to beadsaccommodate all tubes. Mix 1 minute on rotator and then place For example, if there are 10 tubes on magnet rack to collect beads mix together 105 μL 20% SDS, 210 μL 1M NaHCO3 and discard supernatant1.785 mL sterile, distilled water.# Resuspend the bead pellet in 3. For Input tubes (see Section C, step 7), add 200 μl IP μL of Elution Buffer (and set aside at room temperature)until Section E. Vortex 4. Add 100 μL of Elution Buffer to mixeach tube containing the antibody/agarose complex. Mix by flicking tube gently.5. Incubate at room temperature for 15 minutes.==== Reverse cross6. Pellet agarose by brief centrifugation (3000-linking 5000 x g for 1 minute) and recover ChIP DNA ===collect supernatant into new microfuge tubes.7. Repeat steps 4-6 and combine eluates (total volume =200 μL).# Incubate beads from above in a E. Reverse Crosslinks of Protein/DNA Complexes to Free DNA1. To all tubes (IPs and Inputs) add 8 μL 5 M NaCl and incubate at 65°C water bath for '''1 hour''', shake 4-5 hours or vortex every 15 minutes overnight to elute reverse the immunoDNA – Protein crosslinks. After this step the sample can be stored at -bound chromatin from 20°C and the beadsprotocol continued the next day.# Spin at 142. To all tubes,000 rpm in a microfuge at room temperature add 1 μL of RNase A and incubate for 3 30 minutesat 37°C.# Collect the supernatant3. Add 4 μL 0.5M EDTA, which contains the ChIP’d DNA. The tubes can be placed on the magnet 8 μL 1M Tris-HCl and 1 μL Proteinase K to facilitate supernatant recovery.each tube and incubate at 45°C for# Incubate the supernatant containing the ChIP’d DNA in a 65°C water bath overnight to complete the reversal of the formaldehyde cross1-links2 hours.