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 the efficiency of cross-linking. The 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 used, including PBS PMSF 10 µl/ml, aprotinin 1 µl/ml and leupeptin 1 µl/ml).
This section was adapted from protocols kindly provided by Henriette O'Green, Luis G. Acevedo and Peggy J Farnham.
Frozen tissues should be thawed on ice. (This process could take hours depending on the amount of tissue). 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. 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 (between 1-3 mm3)
Determine the weight of an empty 15 ml conical tube, transfer tissue into the tube and weigh again to calculate the amount of tissue
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 minutes
Stop the cross-linking reaction by adding glycine to a final concentration of 0.125 M. Continue to rotate at room temp for 5 minutes
Centrifuge tissue samples for 5 minutes, 720 rpm, 4°C
Aspirate media and wash with 10 ml ice-cold PBS. Centrifuge for 5 minutes, 720 rpm, 4°C and discard wash buffer
*The tissue may be snapped frozen at this stage in liquid nitrogen and stored at -70°C. Avoid multiple freeze-thaws. If using immediately, resuspend tissue in 10 ml cold PBS per gram of starting material. Place on ice
The Medimachine from Becton Dickinson may be used to obtain a single cell suspension. Use 2 medicones (50 um) 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 into 1 ml of PBS
Add this solution to the medicone and grind tissue for 2 minutes
Collect cells from the medicone by inserting an 18 gauge blunt needle and a 1 ml syringe. Collect cells in conical tube on ice
Repeat 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 2.2 to 2.5 until all tissue is ground into a homogeneous suspension
Centrifuge cells for 10 min, 1000 rpm, 4 °C. Measure/estimate cell pellet volume for next step
Carefully aspirate off supernatant and resuspend pellet in FA Lysis Buffer (750 μl per 1x107 cells)