Chromatin preparation from tissues for chromatin immunoprecipitation (ChIP)

This protocol describes how chromatin is prepared from tissue, which can subsequently be used 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:

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.

It is important that the frozen tissue samples do not reach high temperatures to prevent sample degradation by proteases. Samples should be kept on ice at all times and all steps performed quickly to minimize thawing.

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

Transfer tissue into a 15 mL conical tube.

Prepare cross-linking solution in fume hood.

This should be carried out in a fume hood as formaldehyde is harmful.

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

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

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

Avoid multiple freeze-thaws.

The tissue may be snap-frozen at this stage in liquid nitrogen and stored at -70°C.

If using immediately, resuspend tissue in 10 ml cold PBS per gram of starting material then place on ice.

Stage 2 - Tissue degradation

Steps

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

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.

Repeat from step two until all the tissue is processed.

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

If more grinding is necessary, add more PBS to the tissue and repeat steps 2 to 5 until all tissue is ground into a homogeneous suspension

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.