HISTONE CHIP PROTOCOL

Learn the stepwise protocol for Histone chromatin immunoprecipitation (ChIP). It includes cell collection, DNA- Protein Cross-linking, cell lysis, chromatin shearing, and magnetic immunoprecipitation.

Cell Collection And Dna-Protein Cross-Linking

  • 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 CellsTrypsin-EDTA Volume
3 x 1061 mL
1 x 1073 mL
5 x 10715 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 CellsCulture Medium Volume
3 x 1062 mL
1 x 1076 mL
5 x 10730 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.

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.

Quantitative PCR Analysis

Before sequencing the samples, we recommend analyzing the immunoprecipitated 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 immunoprecipitated 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.

ChIP-Seq Protocol

This protocol has been optimized forchip-seq histone modificationl 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.