实验概要
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 ug 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 can be found here and should be used after the chromatin preparation detailed below. Protease inhibitors should be included in all solutions used, including PBS [PMSF (10 ul/ml), aprotinin (1 ul/ml) and leupeptin (1 ul/ml)].
主要试剂
FA Lysis buffer:
50 mM HEPES-KOH pH7.5
140 mM NaCl
1 mM EDTA pH8
1% Triton X-100
0.1% Sodium Deoxycholate
0.1% SDS
Protease Inhibitors (add fresh each time)
实验步骤
1. Cross-linking
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. |
1) Chop frozen or fresh tissue into small pieces using two razor blades (between 1-3 mm3).
2) Determine the weight of an empty 15 ml conical tube, transfer tissue into the tube and weigh again to calculate the amount of tissue.
3) 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.
4) 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.
5) Centrifuge tissue samples for 5 minutes, 720 rpm, 4°C.
6) 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. |
7) If using immediately, resuspend tissue in 10 ml cold PBS per gram of starting material. Place on ice.
2. Tissue disaggregation
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.
1) Cut a 1 ml pipette tip to make the orifice larger.
2) Add between 50-100 mg (3-4 chunks) of tissue resuspended into 1 ml of PBS.
3) Add this solution to the medicone and grind tissue for 2 minutes.
4) Collect cells from the medicone by inserting an 18 gauge blunt needle and a 1 ml syringe. Collect cells in conical tube on ice.
5) Repeat step two until all the tissue is processed.
6) 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.
7) Centrifuge cells for 10 min, 1000 rpm, 4 °C. Measure/estimate cell pellet volume for next step.
8) Carefully aspirate off supernatant and resuspend pellet in FA Lysis Buffer (750 μl per 1x107 cells).
9) Please continue with the X-ChIP protocol from stage two (sonication).
首页
