Native ChIP-seq

Native ChIP using mouse E12.5 heart

Keisuke Nimura
 

・I used eight E12.5 hearts for two samples for ChIP.

・One E12.5 heart includes ~2.5 X 10E5 cardiomyocytes.

・I used 1X10E6 cardiomyocytes for 1 sample.

1. Embryonic hearts are homogenized at 2,000rpm for 5 seconds in a homogenizer (Multi-Beads Shocker, Yasui Kikai) in 1 ml NIB NP40 (complete EDTA free protease inhibitor (11873580001, Roche) and DTT, see below for buffer composition). Incubate sample on ice for 10 minutes in 1.5ml low-binding tube (Cat. BM4015, BMbio).

2. Centrifuge at 10 kg for 1 minute at 4 ˚C. Collect the supernatant in a new tube as cytoplasm fraction.

3. Add 250 µl NIB 400 (complete EDTA free protease inhibitor (Roche) and DTT. See below for buffer composition). Suspend pellet using low-binding tips. Add 1200U MNase (TAKARA, 20U/ul, 40ul), invert 20 times. Incubate sample at 25 ˚C for 30 minutes. Then, incubate the sample at 4 ˚C for 10 minutes.

4. Add 5.5 µl 0.5M EDTA to sample. Incubate on ice for 10 minutes.

5. Centrifuge at 15,000 rpm for 5 minutes at 4 ˚C. Collect supernatant as the nuclear fraction.

6. Add 250 µl NIB 0 (complete EDTA free protease inhibitor (Roche) and DTT. See below for buffer composition). The final concentration of NaCl will be 200 mM. Invert 30 times.

7. Collect 50 µl sample as an input. 

8. Bind antibody to Protein G agarose, Fast Flow (16-266, Millipore). Wash 30 µl beads with 100 µl PBS twice. Add 500 µl PBS and antibody (e.g., 10-15 µg) in the tube. You may make negative control antibody-protein G agarose-conjugate beads. Use ChIP-grade antibody for negative control. Rotate at 4 ˚C for 2 hours. Before using the antibody-conjugating beads, wash the beads with 500 µl PBS twice.

When you centrifuge sample with agarose-conjugate beads, centrifuge sample at 3,000 rpm for 1 second with the protruding tube on the outside -> 3,000 rpm for 1 second with the protruding tube on the inside -> 2,000 rpm for 1 second with the protruding tube on the outside. Then, you can see beads on the bottom of the 1.5 ml tube.

9. Pre-clear sample with, e.g., normal rabbit IgG-AC (sc-2345, Santa Cruz) after washing the beads using 100 µl NIB 200 (complete EDTA free protease inhibitor (Roche) and DTT, see below for buffer composition) twice. Add 1 µl NIB NP40. Rotate at 4 ˚C for more than 1 hour.

10. Centrifuge the pre-clearing sample as described above, collect the supernatant.

11. Add half of the pre-clearing supernatant into the antibody-agarose conjugate beads after removing PBS. Add 1 µl NIB NP40, then rotate sample at 4 ˚C overnight.

12. Wash the beads with 500 µl NIB 500 three times, then 500 µl LiCl buffer 1 time, and then 500 µl TE twice.

13. Add 200 µl ChIP direct elution buffer and 2 µl RNase (2.5 mg/ml), then incubate sample at 37 ˚C for 30 minutes.

14. Add 2 µl Proteinase K (6 mg/ml), then incubate at 55˚C for 1 hour.

15. Add 3 µl Ethachinmate (312-01791, Nippon gene), then vortex. Centrifuge sample, then collect the supernatant.

16. Add 210 µl phenol/chloroform/isoamyl alcohol (25:24:1) (26058-54, Nakalai), then vortex. Centrifuge at 150,000 rpm at room temperature for 10 minutes. Collect supernatant.

17. Add 180 µl TE and 7.2 µl 5 M NaCl into the tube including phenol. Centrifuge at 150,000 rpm at room temperature for 10 minutes. Collect supernatant, then transfer the supernatant into the step 16 tube.

DNA is also extracted with phenol/chloroform/isoamyl alcohol from the input sample, the same as the ChIP sample.

You can use the ChIP DNA Clean and Concentration Kit (D5205, Zymo) instead of phenol/chloroform extraction.

18. Add 900 µl 100% EtOH to the ChIP sample, then incubate the sample at -30 ˚C for more than 1 hour.

19. Centrifuge the sample at 150,000 rpm for 30 minutes at 4˚C.

20. Remove supernatant. Wash the pellet with 1 ml 70% EtOH. 

21. Centrifuge the sample, then remove EtOH. Dry up. Avoid drying up too long.

22. Dissolve the ChIP DNA with 50 µl TE.

You may use 10 times-diluted input for standard in real-time PCR analysis.

Buffer

・NIB NP40: 10mM Tris-HCl pH7.5, 60mM KCl, 1.5mM MgCl₂, 1mM CaCl₂, 0.25M Sucrose, 10%Glycerol, 15mM NaCl, 0.15%NP-40 (add in use. 1mM DTT, Complete EDTA free (Roche) 1/50 vol.)

・NIB 200 : 10mM Tris-HCl pH7.5, 60mM KCl, 1.5mM MgCl₂, 1mM CaCl₂, 0.25M Sucrose, 10%Glycerol, 200mM NaCl, (add in use. 1mM DTT, Complete EDTA free (Roche) 1/50 vol.)

・NIB 400: 10mM Tris-HCl pH7.5, 60mM KCl, 1.5mM MgCl₂, 1mM CaCl₂, 0.25M Sucrose, 10%Glycerol, 400mM NaCl, (add in use. 1mM DTT, Complete EDTA free (Roche) 1/50 vol.)

・NIB 0: 10mM Tris-HCl pH7.5, 60mM KCl, 1.5mM MgCl₂, 1mM CaCl₂, 0.25M Sucrose, 10%Glycerol, 0mM NaCl, (add in use. 1mM DTT, Complete EDTA free (Roche) 1/50 vol.)

・NIB 500: 10mM Tris-HCl pH7.5, 60mM KCl, 1.5mM MgCl₂, 1mM CaCl₂, 0.25M Sucrose, 10%Glycerol, 500mM NaCl,

・ChIP direct elution Buffer: 10mM Tris-HCl pH7.5, 300mM NaCl, 5mM EDTA, 0.5%SDS

・LiCl Buffer : 0.25M LiCl, 0.5% NP40, 0.5% Na-deoxycholate, 1mM EDTA, 10mM Tris-HCl, pH 7.5

Category