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1. Fixation, Cell Lysis, and Chromatin Shearing
NOTE: Patient samples and Jurkat cells are fixed and lysed with a commercially available ChIP kit according to the manufacturer's instructions with modifications as described previously. The fixation is performed under a laminar flow hood.
- Prepare a 1.5 mL tube with 1 mL of 37% formaldehyde, a 1.5 mL tube with 1 mL of 1.25 M glycine, a 5 mL tube with 4 mL of 1x PBS and a box with ice for the fixation of the cells.
- After stimulation of the cells for the respective incubation time, Stimulate the cells by adding 32.4 nM phorbol 12-myristate 13-acetate (PMA) and 1 µM ionomycin. Then incubate the cells for 16 h at 37 °C and 5% CO2 with the lid of the tube opened (to avoid contamination, a sealing film permeable to air can be used, spin the 5 mL tubes at 200 x g for 5 min and resuspend the cells in 500 µL of PBS in the 5 mL tubes.
- Add 340 µM formaldehyde (13.5 µL of 37% formaldehyde) to the cells. After brief mixing by carefully pipetting up and down, incubate the suspension for 2.5 min (Jurkat cells) or 5 min (patient sample) at room temperature.
NOTE: Prolonged fixation time is needed for primary cells to assure optimal shearing.
- Add 125 mM glycine (57 µL of 1.25 M glycine) to stop the fixation and incubate for another 5 min. Put the cells on ice immediately thereafter and centrifuge at 500 x g for 5 min at 4 °C. Remove the supernatant by aspiration.
- Wash the cells twice with 1 mL of ice-cold PBS at 200 x g for 5 min at 4 °C and remove the supernatant every time by aspiration.
NOTE: After the fixation, the protocol can be paused. Fixed cells should be stored at -80 °C. To test, if the epitope of the antibody intended to use in the following protocol is not masked via fixation, additional samples can be used for analysis via SDS-PAGE gel electrophoresis and Western blot. These steps are performed with 100 µg of protein according to manufacturer's instructions. All αNFAT2 antibodies are used at a dilution of 1:1000, the GAPDH antibody at a dilution of 1:10000. An exemplary image of fixed samples from Jurkat cells with appropriate and inappropriate antibodies is shown in Figure 1.
- Resuspend the cell pellet in 5 mL of commercially available lysis buffer 1 by pipetting up and down. Then, place the samples on the ice on a shaker and incubate them for 10 min while shaking.
NOTE: Prior to lysis, the cells can be disintegrated for 10 s in liquid nitrogen. This is useful for Jurkat cells but should be avoided in primary patient samples. For the disintegration, place the 5 mL tubes for 5 s in 5-10 mL of liquid nitrogen in a specific container. Wear safety glasses and appropriate safety gloves.
- Subsequently, centrifuge the tubes at 500 x g for 5 min at 4 °C and remove the supernatant carefully by pipetting.
- Homogenize the cells in 5 mL of commercially available lysis buffer 2 by pipetting up and down and incubate for 10 min on ice while shaking. Then centrifuge the tubes at 500 x g for 5 min at 4 °C and remove the supernatant carefully by pipetting.
- Resuspend the cell pellet in 500 µL (Jurkat cells) or 140 µL (patient samples) of shearing buffer 1 containing 1x protease inhibitor (5 µL or 1.4 µL, respectively) and incubate the mixture for 10 min on ice.
- For chromatin shearing, transfer 140 µL of the cell-suspension from step 1.9 into sonicator tubes (avoid producing air bubbles and repeat this step if necessary due to sample volume). Place the tubes in the focused ultra-sonicator at a temperature of 7 °C and shear for 10 min (cell line) or 7.5 min (patient samples) with an average incident power of 9.375 W to obtain 200-500 bp DNA fragments.
- Finally, centrifuge at 15700 x g for 10 min at 4 °C in the small centrifuge and collect the supernatant in a new 1.5 mL tube.
- To test for the appropriate fragment sizes, analyze 20 µL of the sheared chromatin via gel-electrophoresis in 1.5% TBE agarose gel. An exemplary image of sheared chromatin from Jurkat cells of good and bad quality is shown in Figure 2. At this point, the protocol can be potentially paused. Sheared chromatin should be stored at -80 °C.
