MBD7 ChIP

CHIP PROTOCOL

(Note: at any time, to avoid producing foam when transferring, pipetting) 

Preparation:

1.Premake protein inhibitor cocktail solution (100-fold stock, 2 tablets into 1ml H20), keep it on ice and PMSF (100-fold stock, 100 mM) at room termperature 

2. Premake Glycine solution (0.75g/5mL Distilled Water)

3. Prepare at least 8 ml/each sample M1 solution with mercapto-ethanol 0.7 ul/ml, protein inhibitor, and PMSF

4. Prepare at least 12 ml/each sample M2 solution with mercapto-ethanol 0.7 ul/ml, protein inhibitor, and PMSF

5. Prepare at least 2 ml/each sample M3 solution with mercapto-ethanol 0.7 ul/ml, protein inhibitor, and PMSF

6. Prepare at least 0.5 ml/each sample SDS lysis solution with protein inhibitor and PMSF

7. Prepare at least 5 ml/each sample ChIP dilution solution with protein inhibitor and PMSF

8. Check protein A agarose/salmon sperm DNA (upstate, Cat.#16-157)

9. Check antibody Conc. For GFP Ab (Cat. #632460, Clotech) 1:200 dilution 

For H3K9 (#ab1220, abcam)

For H3K4 (#ab8580, abcam) 

For H3K27 (#07-322, upstate)

10. Check these regular chemicals: EDTA, Tris-HCL, NP-40, LiCi, sodium deoxycholate (#D5670, Sigma), Triton X-100, SDS, NaCl, NaHCO3, mercaptoethanlo, phosphate buffer, hexylene glycol (#M9671, Sigma)

DAY 1 ~4-5 hrs

1. Carefully grind the inflorescences to fine powder (2 ml for two IP) in liquid N₂ and pour the powder into 15 ml yellow tube.

2. Set the tube on ice, add 8 ml M1 buffer and invert several times to mix.

3. Add 216uL formaldehyde (final conc 1%, had better fresh, it's easily oxidized) and incubate on shaker in cold room for 10 min.

4. Add 543uL 2M Glycine (final conc: 0.125M) to stop the crosslink and incubate on shaker in cold room 5 min.

5. Filter the solution through 4-layer of miracloth into a 50 ml yellow tube.

6. Centrifuge at 12,000rpm for 10 min at 4 degree.

7. Discard the supernatant and add 4ml M2 buffer and mix by pipetting. Even out the solution in two 2 ml tubes at this point. Wash 3 times with 1ml M2 and once with 1 ml M3, each wash by spin 1 min at 13200rpm, 4 degree. In the last time, remove all of the solution with a small tip pipette.

SOLUTIONS

M1

10mM phosphate buffer 0.1M NaCl

10mM mercapto-ethanol

1 M hexylene glycol (2-methyl-2-4-pentanediol M9671 Sigma)

M2

10mM phosphate buffer 

0.1M NaCI

10mM mercapto-ethanol 

1M hexylene glycol 

10mM MgC1₂

0.5% Triton-X

M3

10mM phosphate buffer 0.1M NaCl

10mM mercapto-ethanol

Nuclei lysis buffer                                                                         for 5 ml

50 mM Tris-HCI, pH 8.0                                                                 0.25 ml of 1 M

10 mM EDTA                                                                                 100 ul 0.5 M EDTA

1% SDS                                                                                         0.25 ml of 20%

PMSF and protease inhibitors                                                      as in buffer 1

ChlP dilution buffer                                                                      for 10 ml

1.1% Triton X-100                                                                           550 ul of 20%

1.2 mM EDTA                                                                                  24 ul of 0.5 M

16.7 mM Tris - HCI, pH 8.0                                                             167 ul of 1 M

167 mM NaCI                                                                                  334 ul of 5 M

PMSF and protease inhibitors                                                        as in buffer 1

Elution buffer                                                                                for 20 ml

1% SDS                                                                                           1 ml of 20%

0.1 M NaHCO₃                                                                                0.168 g

11. Remove the supernatant and resuspend the chromatin pellet in or 300ul of Nuclei lysis buffer.

12. Resuspend the supernatant by pipetting up and down and gentle vortexing (keep solution cold between vortexing). Keep 1-2ul aliquot to run a gel to compare chromatin to matched sonicated samples.

13. Once resuspended, sonicated the chromatin solution for 10 seconds X 7 times

~15% power (Brason250). Leave it on ice for 20 seconds between each sonication. Following this step, the chromatin solution can be frozen at -20 degrees indefinitely. Note: Supernatant is usually less than 300ul following sonication due to liquid loss. I have been getting~240ul back. Still bring up the volume to 3mls with ChIP dilution buffer in the following steps

14. Spin the chromatin solution for 5 minutes at 4 degrees to pellet debris. Remove supernatant to a new tube. (keep 1-2ul to check sonication efficiency on a gel and compare with aliquot from step 12.)

Note: remove 10ul from each genotype for total DNA control

15. Measure out the remaining volume of sonicated chromatin and bring volume up to 3mls with ChIP dilution buffer.

16. Split the chromatin solution for each genotype 3 ways into tubes (1ml each). Preclear each 1ml of chromatin sample with 40ul protein A Agarose beads for 1hour at 4 degrees with rotation. (Note: prior to use, the beads should be rinsed 3X with ChIP dilution buffer)I prefer to clean the beads in freshly labeled tubes and then split the chromatin solution three ways into those tubes. In this way, you accurately measure the beads. Cut off the end of a tip to measure out the beads).

17. Spin the chromatin solution plus beads at 4 degree for 2 minutes at 10000 rpm.

18. Split solution into new tubes

a. No antibody tube

b. Antibody tubes (add 10ul H3K4 or H3K9 antibodies, concentrations may vary depending on which company do you get these antibodies from)

19. Incubate these tubes with gentle agitation in cold room for several hours minimum, I usually incubate them overnight.

DAY2

20. Add 50uL of Protein A beads (rinsed with ChIP dilution buffer) to each test tube and incubate in cold room shaker for 1 hr.

21. Pellet beads by centrifugation (1 min, 2000rpm, 4 degree).

22. Wash the beads with genitle agitation at 4 degree each wash,then spin at 2000 rpm for 1 min, 4 drgree, with 1 ml of buffer per wash followed by pelleting the beads Apply the following washes in the order listed below:

a. Low salt wash buffer. When wash buffer is added, transfer the beads and solution into a new 1.5mL tube in which the operation is easy and beads could not be lost.

Two washes: one quick, second for 5 minutes.

b. High salt wash buffer. Two washes: one quick, second for 5 minutes.

c. LiCl wash buffer. Two washes: one quick, second for 5 minutes.

d. TE buffer. Two washes: one quick, second for 5 minutes.

23. After the final wash, remove TE thoroughly. Add 250uL Elution buffer (freshly made, 0.084g NaHCO3 + 1 mL 10% SDS + ddH₂O to 10mL), vortex briefly to mix and incubate at 65 degree for 15 min, then centrifuge at 2000 rpm for 1 min and carefully transfer the supernatant (eluate) to a fresh tube and repeat elution of beads. Combine the two eluates.

24. Add 20uL 5M NaCl the eluate and reverse the crosslink at 65 degree at least 6 Note: Do not forget to get your total DNA out of the freezer and reverse X-link with other samples following the same procedure.

DAY3

25. Add 10uL of 0. 5 M EDTA pH 8.0, 20uL IM Tris-HCl, pH 6.8, and 1.5uL of 18.9 mg/mL proteinase K to the 500uL solution and incubate for 1 h at 45 degree. Add 10 uL of 2ug/uL RNAse A to each tube and incubate at RT for 30 min.

26. Recover DNA by phenol/chloroform extraction and EtOH precipitation with 1/10 vol. salt and glycogen. Wash pellets with 70% ethanol.

Note: I often recover DNA by miniprep kit of plasmid DNA (Qiagen). That is, add 1/10v 3M NaAc and 5vol. PB buffer into the solution, then follow the kit procedure.

27. Resuspend the pellet in 100uL of 10mM Tris pH8 (elution buffer replace for kit method). Store them at -20 degree. Usually, I use lul as template in 25uL PCR system.

For formaldehyde, keep freshly, maybe had better store in small aliquots, because it's easily oxidized

Generally, polyclonal antibody is better than monoclonal antibody ProteinA with highaffinity from rabbit polyclonal antibody

ProteinG with highaffinity from rabbit and mouse polyclonal antibody Column method is better than phenol-choloroform method to recover DNA

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