1. Cell Collection and DNA-Protein Cross-Linking (For Cultured Cells)

    The protocol below is intended for adherent cells. For suspension cells, collect them by centrifugation (start with step stated with a * below).

    • Pre-warm PBS, culture medium and trypsin-EDTA at 37°C.
    • Remove the medium and rinse the cells with pre-warmed PBS (10 mL for a 75 cm2 culture flask). Gently shake the flask for 2 min.
    • Remove the PBS and add sterile trypsin-EDTA to the culture flask to detach adherent cells from the bottom. The table below shows the required amount of trypsin for different numbers of cells. Gently shake the culture flask for 1-2 min or until the cells start to detach. The time needed may depend on the cell type.
      Number of Cells Trypsin-EDTA Volume
      3 x 106 1 mL
      1 x 107 3 mL
      5 x 107 15 mL

      Note: Do not continue trypsin treatment longer than necessary as prolonged treatment with trypsin may damage the cells. Regularly check if the cells start to detach.

    • Immediately add fresh culture medium to the cells when they are detached (refer to the table below for volume). This will inactivate trypsin. Transfer cell suspension to a 50 mL tube.
      Number of Cells Culture Medium Volume
      3 x 106 2 mL
      1 x 107 6 mL
      5 x 107 30 mL
    • Rinse the flask by adding 10 mL of PBS. Add this volume to your 50 mL tubes containing cells from the above bullet.
    • * Centrifuge for 5 min at 1,600 rpm and 4°C and remove the supernatant.
    • Resuspend the cells in 20 mL of PBS and count them.
    • Collect the cells by centrifugation for 5 min at 1,600 rpm and 4°C.
    • Resuspend the cells in PBS to obtain a concentration of up to 10 million cells per 500 µL of PBS. If desired, the cell concentration can be decreased to 1 million per 500 µL of PBS.
    • Label 1.5 mL tubes and aliquot 500 µL of cell suspension in each tube.
    • Add 13.5 µL of formaldehyde 37% to each tube containing 500 µL of cell suspension. Mix by gentle vortexing and incubate for 8 min at room temperature to allow fixation to take place.

    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.
    • Add 57 µL of glycine to the cells to stop the fixation. Mix by gentle vortexing and incubate for 5 min at room temperature. Keep the cells on ice from this point onwards.
    • Collect the cells by centrifugation at 1,600 rpm for 5 min and 4°C. Discard the supernatant without disturbing the cell pellet.
    • Wash the cells 2X with 1 mL of cold PBS.
    • Proceed to 3: Cell Lysis and Chromatin Shearing (For Cultured Cells).
  3. Tissue Disaggregation and DNA-Protein Cross-Linking (For Fresh and Frozen Tissue)

    • Weigh 30-40 mg of fresh or frozen tissue in a petri dish. Keep samples on ice at all times and minimize the time of manipulation to prevent sample degradation.
    • Chop tissue into small pieces (between 1-3 mm3) using a scalpel blade.
    • Add 1 mL of ice-cold PBS with protease inhibitor cocktail and disaggregate the tissue using a dounce homogenizer (loose pestle) to get a homogeneous suspension.
    • Transfer the tissue suspension into a 1.5 mL tube and centrifuge at 1,300 rpm for 5 min at 4°C. Gently discard the supernatant and keep the pellet.
    • Resuspend the pellet in 1 mL of PBS containing 1% of formaldehyde at room temperature.
    • Rotate tube for 8-10 min at room temperature.

      Note: The fixation time might require an additional optimization. In general, histone marks require shorter fixation (8 min) than transcriptional factors (10-15 min). Stronger fixation may lead to chromatin resistant to sonication.

    • Stop the cross-linking reaction by adding 100 µL of glycine. Continue to rotate at room temperature for 5 min.
    • Centrifuge samples at low speed (1,300 rpm) at 4°C.
    • Wash the pellet with ice-cold PBS. Aspirate the supernatant and resuspend the pellet in 1 mL of ice-cold PBS plus protease inhibitors.
    • Centrifuge at low speed (1,300 rpm) at 4°C and discard the supernatant.
    • Repeat the washing 1X.
    • Proceed to 4: Cell Lysis and Chromatin Shearing (Derived from Tissue Samples).
  4. Cell Lysis and Chromatin Shearing (For Cultured Cells)

    • Add 1 mL of ice-cold lysis buffer C to the 1.5 mL tube containing 10 million cells. Resuspend the cells by pipetting up and down several times and transfer them to a 15 mL tube. Add 9 mL of lysis buffer C and incubate for 10 min at 4°C with gentle mixing.

      Note: If the starting amount of cells was less than 10 million, scale down accordingly (e.g. Use a total of 5 mL lysis buffer C for 5 million cells).

    • Pellet the cells by centrifugation at 1,600 rpm for 5 min and 4°C and discard the supernatant.
    • Add 1 mL of ice-cold lysis buffer B and resuspend the cells by pipetting up and down several times. Add another 9 mL of lysis buffer B and incubate for 10 min at 4°C with gentle mixing. Scale down accordingly when using less than 10 million cells.
    • 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 B. Prepare 1 mL of complete shearing buffer per tube of 10 million cells. Keep on ice.
    • Add 1 mL of complete shearing buffer B to 10 million cells. Resuspend the cells by pipetting up and down several times. The final cell concentration should be 1 million cells per 100 µL shearing buffer B.
    • Split into aliquots of 100 to 300 µL and transfer the cell suspension to 1.5 mL sonication tubes.
    • Incubate on ice for 10 min. Vortex and spin down the samples.
    • Split the samples into 300 µL aliquots in 1.5 mL sonication tubes and incubate on ice for 10 min.
    • 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 or store it at -80°C for up to 2 months.
    • Proceed to 5: Magnetic Immunoprecipitation.
  5. Cell Lysis and Chromatin Shearing (Derived from Tissue Samples)

    • Add 10 mL of ice-cold lysis buffer C to the pellet corresponding to 30-40 mg of tissue.
    • Resuspend the pellet by pipetting up and down and incubate for 10 min at 4°C with gentle mixing.
    • Centrifuge for 5 min at 1,300 rpm at 4°C and discard the supernatant.
    • Add 10 mL of ice-cold lysis buffer B to the pellet. Resuspend the pellet by pipetting up and down and incubate for 10 min at 4°C with gentle mixing.
    • Centrifuge for 5 min at 1,300 rpm at 4°C and discard the supernatant.
    • Resuspend the pellet in 1.8 mL of shearing buffer B containing protease inhibitors cocktail and homogenize using a dounce homogenizer (tight pestle).
    • Split the samples into 300 µL aliquots in 1.5 mL sonication tubes and incubate on ice for 10 min.
    • Shear 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). Vortexing is not required between runs.
    • Transfer samples to new 1.5 mL tubes and centrifuge at 13,000 rpm for 10 min.
    • Collect the supernatant which contains the sheared chromatin.
    • Store the chromatin at -80°C for up to 2 months for further use in the immunoprecipitation.
  6. Magnetic Immunoprecipitation

    Note: This protocol has been optimized for 1 million cells per ChIP. Although it is possible to use more cells, we recommend performing separate ChIP reactions 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 (20 µL/IP).
    • Dilute the 5X ChIP buffer B with ChIP-seq grade water to obtain 1X ChIP buffer B. The total amount of 1X ChIP buffer B needed is 9X the volume of beads required for the experiment. Place the diluted ChIP buffer on ice.
    • Wash the beads 4X with 2X volume of ice-cold 1X ChIP buffer B. To wash the beads, add 1X ChIP buffer, resuspend the beads by pipetting up and down several times and place the tubes in a suitable magnetic rack for 1.5-mL tubes. Wait for one min to allow the beads to be captured by the magnet and remove the supernatant.
    • Repeat the above 3X. 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.
    • Prepare the ChIP reaction mix according to the table below.
      No. of IPs 5% BSA 200X Protease Inhibitor Cocktail 5X ChIP Buffer B Sheared Chromatin (1x1010 cells) Magnetic Beads ChIP-seq Grade Water Antibody
      1 6 µL 1.5 µL 56 µL 100 µL 20 µL (116.5 – V) µL V µL
      2 12 µL 3.0 µL 112 µL 200 µL 40 µL (233 – V) µL V µL
      4 24 µL 6.0 µL 224 µL 400 µL 80 µL (466 – V) µL V µL
      6 36 µL 9.0 µL 336 µL 600 µL 120 µL (699 – V) µL V µL
      8 48 µL 12.0 µL 448 µL 800 µL 160 µL (932 – V) µL V µL

      Note: If required, sodium butyrate NaBu, a potent deacetylase inhibitor, (20 mM final concentration) or other inhibitors can also be added.

    • Add the antibody to the reaction mix. Use 2 µL of the rabbit IgG control antibody for the negative control IP. If a positive control IP is included in the experiment, use 1 µL of the H3K4me3 ChIP-grade control antibody.
    • When preparing the reaction mix, place 1 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 min 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.
  7. Elution, Decross-Linking and DNA Isolation

    • After removing the last wash buffer, add 400 µL of elution buffer A to the beads and incubate for 30 min under constant rotation at room temperature.
    • Briefly spin the tubes and place them in a magnetic rack.
    • Transfer the supernatant to a new tube and add 16 µL of elution buffer B. Also add 399 µL elution buffer A and 4 µL of elution buffer B to the 1 µL input sample kept aside the day before.
    • Incubate for 4 hours in a thermomixer at 1,300 rpm and 65°C. If required, the incubation at 65°C can be performed overnight.
    • 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.
    • Vortex briefly and perform a short spin.
    • Add 400 µL of 100% isopropanol to each IP and input sample.
    • Vortex briefly and perform a short spin.

      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 15 µL to each IP and input sample (Final volume should be 817 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 1 hour at room temperature on a rotating wheel (40 rpm).
    • Prepare the wash buffer 1 containing 50% isopropanol 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 1.5-mL tubes, wait 1 min and discard the buffer.
    • Add 100 µL wash buffer 1 per tube. Close the tubes, invert the 8-tube strip to resuspend the beads and incubate for 5 min at room temperature on a rotating wheel (40 rpm).

      Note: Do not disturb the captured beads attached to the tube wall. 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 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, invert the 8-tube strip to resuspend the beads and incubate for 5 min at room temperature on a rotating wheel (40 rpm).

      Note: Do not disturb the captured beads attached to the tube wall. 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 1.5-mL tubes, wait 1 min and discard the buffer.
    • Keep the captured beads and add 25 µL of elution buffer C. Close the tubes, invert the 8-tube strip to resuspend the beads and incubate for 15 min at room temperature on a rotating wheel (40 rpm).
    • Resuspend the pelleted beads using the pipet and make sure that you drop them on the bottom of the tube.
    • 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. Keep the bead pellets on ice.
    • Repeat the elution of the bead pellets for 15 min at room temperature on a rotating wheel (40 rpm) in 25 µL elution buffer C.
    • Spin the tubes and place them into a suitable magnetic rack for 1.5-mL tubes, wait 1 min and pool the supernatant with the corresponding IP or input sample (1.5 mL tube). Discard the beads.

      Note: Total elution volume for both IP and input samples is 50 µL (1.5 mL tube).

    • 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.

  8. 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 (GAPDH SS) and negative (Myoglobin exon 2) control primer pair which can be used for the positive control antibody provided in the kit (ChIP-seq grade H3K4me3 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.

  9. ChIP-seq

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