1. Chromatin Immunoprecipitation

NOTE: The chromatin immunoprecipitation was performed with a commercially available ChIP kit according to the manufacturer's instructions with modifications.

1. Calculate the number of immunoprecipitations (70 µL (Jurkat cells) or 15 µL (patient samples) of sheared chromatin plus one antibody) and prepare 20 µL of protein A coated beads per precipitation in a 1.5 mL tube. Wash the beads in 40 µL of 1x ChIP buffer 1 per precipitation by pipetting up and down, let the beads rest in a magnetic rack for 1 min and remove the supernatant by pipetting. Perform this step four times in total. Eventually, resuspend the beads in the original volume with 1x ChIP buffer 1.
2. For the immunoprecipitation itself, use 10 µg of the αNFAT2 antibody (7A6) to capture NFAT2 with its bound target sequences. Use 2.5 µg of a rabbit IgG antibody (DA1E) as an IgG-control. Prepare the reactions in fresh 1.5 mL tubes as indicated in Table 1.
   NOTE: It is important to use a monoclonal antibody with high affinity whose epitope is not masked during fixation for this protocol. Polyclonal antibodies introduce an additional level of variation making it significantly more difficult to appropriately interpret the received results.
3. Incubate the mixture from step 1.2 overnight at 4 °C on a rotating wheel at 6 rpm.
4. On the next day spin the tubes for 5 s at 7,000 x g in the small centrifuge, incubate them in a magnetic rack for 1 min and remove the supernatant by aspiration. Wash the beads once with each of the wash buffers 1, 2, 3 and 4 consecutively.
5. To do so, resuspend the beads in 350 µL of wash buffer and incubate them for 5 min at 4 °C on a rotating wheel at 6 rpm.
6. Thereafter, spin the tubes for 5 s at 7000 x g in the small centrifuge. Then, place them for 1 min in a magnetic rack, remove the supernatant and add the next wash buffer.
7. After the last washing step, remove the supernatant by pipetting, take up the beads in 100 µL of elution buffer 1 and incubate them for 30 min on a rotating wheel at 6 rpm.
8. Spin the tubes shortly (5 s at 7000 x g in the small centrifuge) and place them in a magnetic rack for 1 min.
9. Then transfer the supernatant by pipetting to a new 1.5 mL tube and add 4 µL of elution buffer 2.
10. To create the input control, mix 1 µL of the sheared chromatin with 99 µL of elution buffer 1 and 4 µL of elution buffer 2 (in this setup, there are three tubes per patient: one input control, one IgG-control and one αNFAT2 sample).
11. Incubate the samples for 4 h at 65 °C and 1300 rpm in a 1.5 mL tube shaker. Add 100 µL of 100% isopropanol, vortex and spin the samples for 5 s at 7000 x g in the small centrifuge.
12. Resuspend the available magnetic beads by thoroughly vortexing, add 10 µL of the beads to every sample and incubate for 1 h at room temperature on a rotating wheel at 6 rpm.
13. After the incubation, spin the tubes for 5 s at 7000 x g in the small centrifuge and place them for 1 min in a magnetic rack. Remove the supernatant by aspiration.
14. Resuspend the beads in 100 µL of wash buffer 1. Invert the tubes to mix and incubate for 5 min at room temperature on a rotating wheel at 6 rpm.
15. Spin the tubes for 5 s at 7000 x g in the small centrifuge, place them for 1 min in a magnetic rack and remove the supernatant by pipetting. Repeat the steps 2.13-2.14 with wash buffer 2.
16. Subsequently, remove the wash buffer by aspiration, resuspend the beads in 55 µL of the elution buffer and incubate for 15 min at room temperature on the rotating wheel at 6 rpm to elute the precipitated DNA. Finally, spin the tubes for 5 s at 7000 x g in the small centrifuge, place them for 1 min in a magnetic rack and transfer the supernatant into new 1.5 mL tubes.
    NOTE: Here the protocol can be potentially paused. The eluted DNA should then be stored at -20 °C.

Table 1: Schedule for pipetting the chromatin immunoprecipitation. The chromatin immunoprecipitation was performed by preparing the reactions as indicated in the table.