Chromatin Immunoprecipitation Protocol ChIP Protocol

1. Resuspend magnetic or agarose Protein A, G, or A/G beads on a rotator at room temperature for 5 minutes.
2. Move required volume of beads to 2 fresh 1.5 ml tubes, transferring 50 μl per sample to each tube. This includes 50 μl for pre-clearing, and 50 μl for immunoprecipitation. Pre-clearing is optional but highly recommended if using agarose beads.
3. For the pre-clearing tube, pellet beads and aspirate supernatant. Replenish with the same volume of ChIP dilution buffer (50 μl per sample) to wash. Repeat this step twice. These beads are now ready and should be stored at 4°C until use.
4. For the immunoprecipitation tube:
1. Wash the beads with 1 ml of bead-binding buffer. Pellet the beads, aspirate the supernatant and repeat.
2. Resuspend beads in bead-binding buffer, 200 μl per IP to be performed.
3. Add 1-10 μg of primary antibody. This amount will need to be optimized for each antibody. There should be a tube for the target protein antibody, and a tube for an isotype control (mock) antibody.
4. Incubate the bead-antibody complexes overnight with gentle rotation at 4°C.
5. Wash the beads twice with 1 ml of bead-binding buffer, and then twice with ChIP dilution buffer.
6. Pellet the beads and aspirate the buffer. Resuspend in 50 μl ChIP dilution buffer per IP to be performed.
7. Store beads at 4°C until the chromatin has been prepared.

Before starting:

• Cool sonicator to 2°C before fragmentation
• Set bench top centrifuge to 4°C
• Use DNA low bind tubes throughout protocol
• Prepare all of your buffers in advance with protease inhibitors
• Keep all buffers on ice

Cell Culture

1. Grow cells to 80-90% confluence in 150 mm culture dishes containing sufficient (~20 ml) culture media.
1. This should provide 1 to 5 x 10⁷ cells
2. Be sure to have enough plates for all conditions (e.g. replicates, treated and untreated etc.).
3. Include one extra plate of cells for the estimation of cell number
2. If needed, treat or stimulate cells with appropriate inducing agent.
3. Add 37% formaldehyde dropwise for a final concentration of 1% (~550 µl formaldehyde to 20 ml media) directly to the growth media to crosslink the cells.
4. Incubate for ~15 minutes (time should be determined empirically) with gentle rotation at room temperature.
5. Add glycine to a final concentration of 0.125 M to each dish to quench the formaldehyde.
6. Mix and incubate for 5 minutes at room temperature.
7. Place dishes on ice and carefully aspirate media. Wash twice with ice-cold PBS.
8. Add 5 ml ice-cold PBS containing protease inhibitors and scrape cells off dish. Transfer to a 50 ml tube.
9. Centrifuge for 5 min, 1000 x g at 4°C.
1. At this point, move on to cell lysis or store the pellet at -80°C for later use.

Tissue

1. Isolate fresh tissue as required, keeping on ice as much as possible.
2. Excise a pea-sized piece of tissue and cut into small chunks (<1 mm), then transfer to a 50 ml tube.
3. Wash twice in ice-cold PBS.
4. Suspend in 20 ml ice-cold PBS and add 550 μl of 37% formaldehyde for a final concentration of 1%.
5. Gently mix and incubate at room temperature for 10 minutes to crosslink.
6. Add glycine to a final concentration of 0.125 M to each tube to quench the formaldehyde.
7. Homogenize the tissue using a Dounce homogenizer.
8. Centrifuge for 5 min, 1000 x g at 4°C.
1. At this point, move on to cell lysis or store the pellet at -80°C for later use.

1. Carefully remove supernatant and resuspend pellet in 6 volumes of ice-cold ChIP cell lysis buffer on ice by pipetting up and down or flicking the tube.
2. Incubate on ice for 20 minutes.
1. This incubation period will result in swelling of cells but keep nuclei intact.
3. Homogenize with a Dounce homogenizer (~30 strokes).
4. Transfer homogenate to a fresh 1.5 ml tube.
5. Centrifuge for 5 min, 2000 x g at 4°C to pellet the nuclei.
1. This pellet should be mostly white and will be looser than the cell pellet was.
6. Aspirate the supernatant and discard. Resuspend the nuclear pellet in 5 volumes (based on original cell pellet volume) of ice-cold ChIP nuclear lysis buffer.
7. Incubate on ice for 20 minutes.

1. Place tubes into bath sonicator set at 2°C.
1. Probe sonicators can also be used. Ensure samples are kept cold to prevent damage to the chromatin.
2. Sonicate the samples to produce DNA fragment sizes of 200-750 bp.
1. This step is highly dependent on cell type, cell concentration and the specific sonicator being used. It will need optimizing prior to the experimental run. To optimize:
1. Try several sonication conditions while keeping the cell number consistent. For example, alter the total sonication time, the pulse width and the rest period between pulses.
2. After trying multiple conditions, 5 µl of the sample can be taken and added to 45 µl elution buffer to run on a DNA gel.
3. Add 10 μg of RNase A for 15 min at 37°C
4. Add 20 μg Proteinase K and incubate at 65°C for 30 min.
5. Reverse crosslinks by incubating at 95°C for 5 minutes, then allow samples to cool slowly to room temperature.
6. Load 10 μl and 20 μl (in case of under- or over-loading) on a 1% agarose gel alongside a DNA ladder.
7. Determine which condition gives a smear of DNA in the target range of 200-750 bp.
8. Modify and repeat optimization if none of the conditions resulted in optimal DNA fragmentation.
9. The volume and cell concentration used in experiments should be kept consistent across samples to ensure fragmentation consistency. If samples end up being larger, divide them between multiple 1.5 ml tubes.
3. Incubate samples on ice for 10 minutes.
4. Centrifuge samples at max speed at 4°C for 10 minutes to remove insoluble material.
5. Keep the supernatant, which contains soluble, sheared chromatin, carefully transferring to a new tube while leaving residual debris. Pool chromatin that was originally from the same sample, if appropriate.
1. 5 μl can be removed from each sample at this point, treated with RNase A, proteinase K and heated as above, followed by DNA purification on a spin column, in order to determine DNA concentration in the samples using a NanoDrop spectrometer or similar.
2. Sheared chromatin can be used immediately or stored at -80°C.

1. Dilute 30-50 μg of chromatin with ChIP dilution buffer in a fresh tube for a final volume of 450 μl.
1. Diluting the chromatin will also reduce the SDS concentration, ideally to <0.1%, as this can affect antibody performance.
2. The amount of chromatin to be assayed is highly antibody- and target-dependent. For example, histones are highly abundant compared to transcription factors. 1-50 μg is a general guide, but the exact amount should be determined empirically for each target-antibody combination.
2. Retain 1-10% of the volume of diluted chromatin as the input material and store at 4°C for later use.
3. Add 50 μl prepared pre-clearing beads to each tube.
4. Incubate with gentle rotation at 4°C for 2 hours.
5. Pellet the beads and transfer the supernatant to a fresh tube.
6. Add 50 μl prepared antibody-beads to each tube to capture protein-DNA complexes.
7. Incubate overnight with gentle rotation at 4°C.

1. Pellet the beads and aspirate the supernatant.
2. Wash 1x with gentle rotation in 1 ml low salt wash buffer for 5 minutes at room temperature.
3. Wash 1x with gentle rotation in 1 ml high salt wash buffer for 5 minutes at room temperature.
4. Wash 1x with gentle rotation in 1 ml LiCl wash buffer for 5 minutes at room temperature.
5. Wash 3x with gentle rotation in 1 ml TE buffer for 5 minutes at room temperature.
6. Pellet the beads and aspirate the supernatant.
7. Add 250 μl elution buffer to the beads, and incubate with rotation for 15 minutes at room temperature.
8. Pellet the beads and transfer the supernatant to a fresh tube, Repeat the elution step on the beads, and pool the eluates (500 μl total volume).
9. Add 80 μl Reverse Crosslinking Buffer to samples. Mix well and incubate at 65º C for at least 5 hours or overnight to reverse crosslinking and degrade proteins and RNA.
10. Purify the DNA using a standard PCR purification kit.
1. Note that the addition of 3 M sodium acetate may be required to adjust the pH for efficient DNA extraction.
11. The DNA is now ready for quantification (e.g. by NanoDrop), PCR, qPCR or library preparation for sequencing, or can be stored at -20°C for later use.