Chromatin preparation from tissues for chromatin immunoprecipitation (ChIP)

Last edited Tue 31 Jan 2023

It is recommended that 30 mg of liver tissue is used for each ChIP/antibody. However, this amount may vary for other tissues. The exact amount of tissue depends upon protein abundance, antibody affinity, and cross-linking efficiency.

This protocol was optimized using 5-15 µg chromatin for each ChIP assay. The exact chromatin concentration should be determined for each tissue type before starting the X-ChIP assay. Our cross-linking chromatin immunoprecipitation (X-ChIP) protocol should be used after the chromatin preparation detailed below. Protease inhibitors should be included in all solutions, including PBS PMSF 10 µL/mL, aprotinin 1 µL/mL and leupeptin 1 µL/mL.

Materials:

• FA lysis buffer
• 50 mM HEPES-KOH pH7.5
• 140 mM NaCl
• 1 mM EDTA pH 8
• 1% Triton X-100
• 0.1% Sodium deoxycholate
• 0.1% SDS
• Protease inhibitors (add fresh each time)

Adapted from protocols kindly provided by Henriette O’Geen, Luis G. Acevedo and Peggy J. Farnham.

Stage 1 - Cross-linking

Steps

Frozen tissues should be thawed on ice.

• This process can take hours, depending on the amount of tissue.
• Tissue should be cut in a petri dish resting on a block of dry ice.

Chop frozen or fresh tissue into small pieces using two razor blades.

• The piece should measure between 1-3 mm³.

Transfer tissue into a 15 mL conical tube.

• Weight the empty tube before and after tissue transfer to calculate the amount of tissue available.

Prepare cross-linking solution in fume hood.

• Use 10 ml PBS per gram of tissue.
• Add formaldehyde to a final concentration of 1.5 % and rotate tube at room temperature for 15 mins.

Stop the cross-linking reaction by adding glycine to a final concentration of 0.125 M.

• Continue to rotate at room temp for 5 mins.

Centrifuge tissue samples for 5 minutes at 100 x g at 4°C.

Aspirate media and wash with 10 ml ice-cold PBS.

• Centrifuge for 5 minutes at 100 x g at 4°C and discard the wash buffer.

Stage 2 - Tissue degradation

Steps

The Medimachine from Becton Dickinson can be used to obtain a single cell suspension.

• Use 2 medicons (50 μm) per gram of tissue to process.

Cut a 1 ml pipette tip to make the orifice larger.

Add between 50-100 mg (3-4 chunks) of tissue resuspended in 1 ml of PBS.

Add this solution to the medicon and grind tissue for 2 minutes.

Collect cells from the medicon by inserting an 18 gauge blunt needle and a 1 ml syringe.

• Collect cells in a conical tube on ice.

Repeat from step two until all the tissue is processed.

Check the cell suspension using a microscope to ensure a unicellular suspension is obtained.

Centrifuge cells for 10 minutes at 300 x g at 4 °C.

Measure/estimate cell pellet volume for the next step.

Carefully aspirate off supernatant and resuspend pellet in FA lysis buffer.

• We recommend 750 μl of lysis buffer per 1x10⁷ cells.