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).
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 (example P408676, P301906), aprotinin 1 µl/ml (example A105534) and leupeptin 1 µl/ml (example L420424).
Materials:
- FA lysis buffer
- 50 mM HEPES-KOH pH7.5 (example H301907)
- 140 mM NaCl
- 1 mM EDTA pH8 (example E196386)
- 1% Triton X-100 (example T434386)
- 0.1% Sodium deoxycholate (example S104198)
- 0.1% SDS (example S108346)
- Protease inhibitors (add fresh each time)
Stage 1 Cross-linking
Steps
1、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.
Warning: 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.
2、Chop frozen or fresh tissue into small pieces using two razor blades.
The piece should measure between 1-3 mm3.
3、Transfer tissue into a 15 ml conical tube.
Weight the empty tube before and after tissue transfer to calculate the amount of tissue available.
4、Prepare cross-linking solution in fume hood.
4.1 Use 10 ml PBS (example T494526) per gram of tissue.
4.2 Add formaldehyde to a final concentration of 1.5% and rotate tube at room temperature for 15 mins.
Warning: This should be carried out in a fume hood as formaldehyde is harmful.
5、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.
6、Centrifuge tissue samples for 5 minutes at 720 rpm at 4°C.
7、Aspirate media and wash with 10 ml ice-cold PBS (example T494526).
Centrifuge for 5 minutes at 720 rpm, at 4°C and discard wash buffer.
Warning: Avoid multiple freeze-thaws.
Notice: 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
1、The Medimachine from Becton Dickinson can be used to obtain a single cell suspension.
Use 2 medicones (50 um) per gram of tissue to process.
2、Cut a 1 ml pipette tip to make the orifice larger.
3、Add between 50-100 mg (3-4 chunks) of tissue resuspended in 1 ml of PBS (example T494526).
4、Add this solution to the medicone and grind tissue for 2 minutes.
5、Collect cells from the medicone by inserting an 18 gauge blunt needle and a 1 ml syringe.
Collect cells in a conical tube on ice.
6、Repeat from step two until all the tissue is processed.
7、Check the cell suspension using a microscope to ensure a unicellular suspension is obtained.
Notice: If more grinding is necessary, add more PBS (example T494526) to the tissue and repeat steps 2 to 5 until all tissue is ground into a homogeneous suspension.
8、Centrifuge cells for 10 mins at 1000 rpm at 4°C.
Measure/estimate cell pellet volume for the next step.
9、Carefully aspirate off supernatant and resuspend pellet in FA lysis buffer.
We recommend 750 μl of lysis buffer per 1x107 cells.
10、From the ultrasonic processing step, proceed to the X-ChIP protocol.
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