ChIP PROTOCOL

Chromatin immunoprecipitation (ChIP) is a technique for studying the interaction of proteins with chromatin DNA, which can be used to identify both interacting proteins and interacting DNA, depending on the purpose.It is often used in epigenetic studies, such as the detection of transcriptional regulation-associated histone modifications by ChIP.

Operational steps:

I. Sample Preparation (Keep samples on ice or at 4°C.)

For suspension cells, ensure that there are an appropriate number of cells in 10 ml of fresh medium and add 37% formaldehyde directly to a final concentration of 1%. After agitation and mixing, incubate for 10 minutes at room temperature on a shaker.

For tissues, grind the tissue well with liquid nitrogen until powdered. Transfer to a 15 ml or 50 ml sharp-bottomed tube and add 10 ml PBS and 270 μL 37% formaldehyde to a final concentration of 1%. After shaking and mixing, incubate for 10 minutes at room temperature on an orbital shaker.

1. The reaction was terminated by the addition of 500 ul of 2.5 M glycine (to a final concentration of 0.125 M) and incubated for 5 minutes at room temperature.

2. Cells were transferred to 50 ml pointed-bottom tubes and centrifuged at 4°C for 5 minutes at 2500 rpm.

3. The supernatant was discarded and the cells were washed twice with ice-cooled PBS (pH 7.4) and centrifuged at 4°C for 5 minutes at 2500 rpm after each wash.

4. Cell lysates were precooled with 1 mL of ice, protease inhibitor was added, and cells were resuspended. To promote cell membrane rupture, cells were homogenized 5 to 10 times with a Douncer glass homogenizer and incubated at 4°C for 15 minutes.

5. Centrifuge at 4000 rpm for 5 minutes at 4°C and discard the supernatant.

6. Cell lysates were renucleated (100 ul of lysate per 3-5 x 106 cells).

7. Sonication on ice was performed for 5-15 min (25% power, 20 s sonication and 30 s pause), and optimal sonication conditions must be determined based on preexperimentation.

8. Centrifuge at 4°C for 5 minutes at 12000 rpm, transfer the supernatant to a new EP tube and perform the following assay immediately or freeze at -20°C as a backup.

II. Detection of DNA Breakage Effects

1. Take 50 ul of chromatin supernatant, add 150 ul ddH2O, 10 ul 5M NaCl, 65 ℃ for 4 h or overnight to uncrosslink.

2. RNaseA was added to a final concentration of 50 μg/mL and incubated at 37°C for 30 minutes.

3. Proteinase K was added to a final concentration of 50 μg/mL and incubated at 42°C for 30 minutes.

4. Kit for purification of DNA fragments.

5. 1.5% agarose gel electrophoresis assay.

III. Immunoprecipitation (by single CHIP reaction)

1. Prepare 70 ul of magnetic beads.

2. The magnetic beads were washed twice with 600 μL PBS containing 5% BSA.

3. After the second wash, resuspend the magnetic beads to the original volume.

4. Take 100 ul of cell lysate (containing approximately 20 ug of chromatin, depending on the cells and sample preparation) and dilute 1:10 to 1 ml.

5. Take 25 ul of washed magnetic beads and add to cell lysates for lysate preadsorption treatment.

6. To the remaining 45 ul of magnetic beads, add 2-5 ug of the appropriate antibody, spin and incubate overnight at 4°C. (Use non-specific IgG as a negative antibody control).

7. Sample from step 5, separate magnetically, and transfer the pre-adsorbed lysate to a new EP Tube.

8. After step 6, wash the antibody-magnetic bead complex 2 times with 300 ul of ice-cooled PBS (containing 5% BSA) and discard the supernatant.

9. Add pre-adsorbed lysate (reserve 50 ul for freezing) to the antibody-magnetic bead complex and incubate for 2 hours at 4°C with rotation.

10. The samples were temporarily removed and then placed on a magnetic stand.

11. 1 mL of low salt wash solution at a time for 2 washes.

12. 1 mL of high salt wash solution at a time, 2 washes.

13. 1 ml LiCl wash solution at a time, washed twice.

14. 1 ml of TE solution at a time and wash 2 times. For the 2nd wash, transfer the sample to a new EP tube.

15. At the end of the final wash, drain the wash solution from the pipette.

16. Prepare Elution Buffer and set the thermostatic bath to 65 ℃.

17. Add 100 ul of Elution Buffer to each sample (after step 15) and incubate at 65°C for 10 minutes.

18. Transfer the eluate to a new EP tube and repeat step 17 once for a final collection of 200 ul.

19. Take 50 ul of lysate as 5% input after pre-adsorption. Add 150 ul of elution buffer to give a total volume of 200 ul.

20. Add 10 ul of NaCl (5 M Masterbatch) to the CHIP-eluted sample (from step 18) or Input and incubate overnight at 65°C to uncrosslink.

IV. DNA purification and analysis:

1. Proteinase K was added to a final concentration of 50 μg/mL and incubated at 42°C for 1 hour.

1. The kit purified the DNA fragments.

2. PCR Detection and Analysis.