1. Cell Collection and DNA-Protein Cross-Linking

    • Dilute formaldehyde in fixation buffer to a final concentration of 11% (e.g. Add 5 mL of a 37% formaldehyde solution to 11.8 mL fixation buffer. For a T175 culture flask you will need ~2 mL of diluted formaldehyde.).
    • Add 1/10 volume of the diluted formaldehyde directly to the cell culture medium.
    • Incubate the cells for 10 to 20 minutes at room temperature with gentle shaking. The fixation time can depend on your target of interest.
    • Add 1/10 volume of glycine to the cell culture medium to stop the fixation. Incubate for 5 min at room temperature with gentle shaking.

    Note: The fixed cells can be stored at -80°C for up to 4 months. However, we strongly recommend using freshly fixed cells for preparation of sheared chromatin prior to ChIP for ChIP-seq.

  2. Cell Lysis and Chromatin Shearing

    (A) Cell Lysis – Part I

    (i) For adherent cells (~25 million cells)

    • Remove the medium and wash the cells 1X with 20 mL of PBS. Keep everything at 4°C from now on.
    • Add 5 mL of cold lysis buffer A to the plate and collect the cells by scraping.
    • Add an additional volume of lysis buffer A to rinse the flask and add this to the collected cells. The total volume of lysis buffer A should be about 10 mL per 107 cells [e.g. For a T175 culture flask (~25 milllion cells), rinse with an additional 20 mL of buffer A].
    • Incubate at 4°C for 20 minutes.

    (ii) For suspension cells (~25 million cells)

    • Pellet the cells by centrifugation at 1,600 rpm and 4°C for 5 minutes. Discard the cell culture medium.
    • Wash the cells once with PBS. Resuspend the cells in 20 mL of PBS, centrifuge at 1,600 rpm and 4°C for 5 min and discard the supernatant. Keep everything at 4°C from now on.
    • Add 1 mL ice-cold lysis buffer A to the cell pellet and resuspend the cells by pipetting up and down several times. Add an additional amount of buffer A to obtain a total volume of about 10 mL per 107 cells [e.g. For a T175 culture flask (~25 milllion cells), add an additional 24 mL of buffer A].
    • Incubate at 4°C for 20 minutes.

    (B) Cell Lysis – Part II

    • Pellet the cells by centrifugation at 1,600 rpm for 5 min and 4°C and discard the supernatant.
    • Add 1 mL ice-cold lysis buffer B to the cell pellet and resuspend the cells by pipetting up and down several times. Add an additional amount of buffer B and incubate for 10 min at 4°C with gentle mixing. For 25 million cells, the total amount of buffer B should be 15 mL.
    • Pellet the cells again by centrifugation for 5 min at 1,600 rpm (500 x g) and 4°C and discard supernatant.
    • Add 200X protease inhibitor cocktail to shearing buffer A. Prepare 1 mL of complete shearing buffer per tube of 15 million cells. Keep on ice.
    • Add 1 mL of complete shearing buffer to 15 million cells. Resuspend the cells by pipetting up and down several times. The final cell concentration should be 1.5 million cells per 100 μL shearing buffer. Split into aliquots of 100 to 300 μL and transfer the cell suspension to 1.5 mL sonication tubes.

    Chromatin Shearing

    • Shear the chromatin by sonication (Optimization on shearing conditions, e.g. number of runs, number of cycles per run, may be required, depending on the sonicator and cell types used).
    • Briefly vortex and spin between each run.
  3. Magnetic Immunoprecipitation

    • Centrifuge at 13,000 rpm (16,000 x g) for 10 min and collect the supernatant which contains the sheared chromatin. Use the chromatin immediately in immunoprecipitation (IP) or store it at -80°C for up to 2 months.

      Note: This protocol has been optimized for ~4 million cells per ChIP, although it is possible to reduce or increase the amount of cells. For using lower amounts of cells, simply dilute the chromatin in shearing buffer A before adding it to the IP reaction. For higher cell numbers you can increase the cell concentration in the shearing buffer, although this may require an additional optimization of the shearing conditions. Therefore, we recommend performing separate ChIPs and pool the samples before purification of the DNA.

    • Determine the total number of IPs in the experiment. We recommend to include one negative control in each experiment (IP with the IgG negative control). Take the required amount of protein A-coated magnetic beads (30 μL/IP).
    • Prepare 4 mL of 1X ChIP buffer A by adding 3.2 mL ChIP-seq grade water to 0.8 mL 5X ChIP buffer A. Add 80 μL of 5% BSA. Keep the 1X ChIP buffer A on ice.
    • Wash the beads 3X with 1 mL of ice-cold 1X ChIP buffer A. To wash the beads, add 1X ChIP buffer, resuspend the beads by pipetting up and down several times and incubate at 4°C with gentle shaking for 5 min. Spin the tubes and place them in a magnetic rack. Wait for one min to allow the beads to be captured by the magnet and remove the supernatant.
    • Repeat the above 2X. Alternatively, centrifuge the tubes for 5 min at 1,300 rpm, discard the supernatant and keep the bead pellet.
    • After the last wash, resuspend the beads in the original volume of 1X ChIP Buffer.
    • Take the required number of tube and add 30 μL of the washed beads to each tube.
    • Prepare the ChIP reaction mix according to the table below.
      No. of IPs 5% BSA 200x Protease Inhibitor Cocktail ChIP Buffer A 5X ChIP-seq Grade Water Antibody
      1 6 μL 1.8 μL 20 μL (42.2 – V) μL V μL
      2 12 μL 3.3 μL 40 μL (84.4 – V) μL V μL
      4 24 μL 7.2 μL 80 μL (168.8 – V) μL V μL
      6 36 μL 10.8 μL 120 μL (253.2 – V) μL V μL
      8 48 μL 14.4 μL 160 μL (337.6 – V) μL V μL

      Note: If you will use different amounts of antibody (e.g. when performing a titration curve), we recommend to add the water separately. Also, if required, sodium butyrate NaBu, a potent deacetylase inhibitor, (20 mM final concentration) or other inhibitors can also be added.

    • Add 70 μL of ChIP reaction mix to the tubes containing the beads.
    • Add the antibody to the reaction mix. Use 1 μL of the IgG negative control antibody for the negative control IP. If a positive control IP is included in the experiment, use 0.5 μL of the CTCF positive control antibody.
    • Incubate the tubes for 2-4 hours at 4°C under constant rotation at 40 rpm.
    • Briefly spin the tubes and add 250 μL of sheared chromatin. Put 2.5 μL of the sheared chromatin aside to be used as an input the next day. Incubate the tubes overnight at 4°C under constant rotation at 40 rpm.
    • The next morning, after the overnight incubation, briefly spin the tubes and place them in a magnetic rack. Wait for one minute and remove the supernatant.
    • Wash the beads by adding 350 μL of wash buffer A, gently shaking the tubes to resuspend the beads, and incubating for 5 min at 4°C under constant rotation at 40 rpm.
    • Repeat the wash as described above 1X with wash buffers B, C and D, respectively.
  4. Elution, Decross-Linking and DNA Purification

    • After removing the last wash buffer, add 100 μL of elution buffer A to the beads and incubate for 30 min under constant rotation at room temperature.
    • Resuspend the beads pellet and transfer it into a new 200 μL tube.
    • Briefly spin the tubes and place them into a magnetic rack.
    • Transfer the supernatant to a new tube and add 4 μL of elution buffer B. Also add 97.5 μL buffer A and 4 μL of buffer B to the 2.5 μL input sample. Incubate for 4 hours or overnight at 65°C.
    • Pool samples if necessary.

      Note: Up to two samples can be easily pooled. If more than two samples need to be pooled, process each sample purification individually, pool final eluates at the end of the magnetic bead purification and concentrate.

    • Add 2 μL of carrier to each IP and input sample.
    • Add 100 μL of 100% isopropanol to each IP and input sample.

      Note: Following the addition of isopropanol the solution may become cloudy. This is not detrimental to your experiment and will not influence the quality or quantity of your purified DNA.

    • Resuspend the high-DNA recovery magnetic beads and transfer 10 μL to each IP and input sample (Final volume should be 116 μL per reaction).

      Note: Keep the beads in liquid suspension during storage at 4°C and at all handling steps, as drying will result in reduced performance.

    • Incubate IP and input samples for 10 min at room temperature on a rotating wheel (40 rpm).
    • Prepare the wash buffer 1 containing 50% isopropanol (for 24 reactions) by mixing 2 mL wash buffer 1 w/o isopropanol and 2 mL isopropanol (100%). Never leave the bottle open to avoid evaporation.
    • Briefly spin the tubes, place them in a suitable magnetic rack for 0.2-mL tubes, wait 1 min and discard the buffer.
    • Add 100 μL wash buffer 1 per tube. Close the tubes and vortex well until the beads pellet is completely broken.

      Note: In order to avoid the beads pellet to be too difficult to break down, do not let the beads for too long on the magnet, incubate for 30 sec at room temperature on a rotating wheel (40 rpm). Always briefly spin the tubes to bring down liquid caught in the lid prior to positioning into the magnetic rack.

    • Prepare the wash buffer 2 containing 50% isopropanol as follows (for 24 reactions) by mixing 2 mL wash buffer 2 w/o isopropanol and 2 mL isopropanol (100%). Never leave the bottle open to avoid evaporation.
    • Briefly spin the tubes, place in a suitable magnetic rack for 1.5-mL tubes, wait 1 min and discard the buffer.
    • Add 100 μL wash buffer 2 per tube. Close the tubes and vortex well until the beads pellet is completely broken.

      Note: In order to avoid the beads pellet to be too difficult to break down, do not let the beads for too long on the magnet, incubate for 30 sec at room temperature on a rotating wheel (40 rpm). Always briefly spin the tubes to bring down liquid caught in the lid prior to positioning into the magnetic rack.

    • Briefly spin the tubes and place them a suitable magnetic rack for 0.2-mL tubes, wait 1 min and discard the buffer.
    • Spin the tubes again and place them on the magnetic rack. Discard the remaining wash buffer 2 if necessary.
    • Resuspend the beads pellet with 25 μL of elution buffer C. Incubate at room temperature for 15 min on a rotating wheel (40 rpm).
    • Spin the tubes and place them into a suitable magnetic rack for 1.5-mL tubes, wait 1 min and transfer the supernatants into a new labelled 1.5 mL tube and discard the beads.
    • Place the DNA on ice and proceed to any desired downstream applications or store it at -20°C or -80°C until further use.
    • Take 5 μL (10%) of immunoprecipitated DNA and determine the concentration with, for example, the Quant-iT High Sensitivity dsDNA Assay Kit and Qubit fluorometer.
    • Store the DNA at -20°C until you are ready to analyze it with qPCR or by high throughput sequencing.

      Note: The elution buffer C is suitable for direct qPCR analysis, whole genome amplification, chip hybridization and Next-Generation sequencing.

  5. Quantitative PCR Analysis

    Before sequencing the samples, we recommend analyzing the immunopreciptated DNA by qPCR using at least one positive and one negative control target. The kit contains a positive (H19 imprinting control region) and negative (Myoglobin exon 2) control primer pair which can be used for the positive control antibody provided in the kit (ChIP-seq grade CTCF antibody) in SYBR Green qPCR assay using the protocol described below. Use your own method of choice for analyzing the appropriate control targets for your antibodies of interest.

    • Prepare the qPCR mix as follows (20 μL reaction volume using the provided control primer pairs):
      • 10 μL of a 2x SYBR Green qPCR master mix
      • 1 μL of primer mix
      • 4 μL of water
      • 5 μL immmunoprecitated or input DNA
    • Carry out the PCR with this sequence:
      • Denaturation step: 95°C (3 to 10 min)
      • 40 cycles of 95°C (30 sec), 60°C (30 sec) and 72°C (30 sec).

        Note: Check carefully supplier’s recommendations about Taq polymerase activation time. These PCR conditions may require optimization depending on the type of Master Mix or qPCR system used.

  6. ChIP-seq

    This protocol has been optimized for ChIP-seq on an Illumina HiSeq sequencer. However, other sequencing systems such as the Illumina MiSeq or the Thermo Fisher SOLiD systems can also be used. Refer to their sequencing protocols for the generation of sequencing data.