ChIP protocol for Transcription Factors

Cell Lysis And Chromatin Shearing

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 million 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). Tissue Disaggregation and DNA-Protein Cross-Linking (For Fresh and Frozen Tissue)
  

  (ii) 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 the 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 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).

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 the 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.

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 into 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.

• Note: If you will use different amounts of antibody (e.g. when performing a titration curve), we recommend adding 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 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.

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.