Micrococcal Nuclease (MNase) Fractionation and Purification of Chromatin

Preparation of Dialysis Tubing (2 h including cooling time)

17. Cut the tubing into pieces of convenient length (10-20 cm).

18. Boil the tubes for 10 minutes in 0.5 liter of tubing preparation solution I.

19. Rinse the tubes twice in distilled H₂O.

20. Boil the tubes for 10 minutes in 0.5 liter of tubing preparation solution II.

21. Allow the tubes to cool down, and store them in tubing preparation solution II at 4°C. Ensure that the tubes are entirely submerged.

22. Before use, wash the tubing twice, inside and out, with H₂O.
Several batches of dialysis tubing can be prepared and stored at 4°C for several weeks.

MNase Fractionation (30 min)

23. Aliquot the resuspended nuclei (from Step 8 or 16) into two 1.5-ml microcentrifuge tubes (500 µl in each tube).

24. Add 1 µl of MNase enzyme (10 units/µl in 50% [v/v] glycerol) to each tube, and mix gently.

25. Incubate the two tubes in a 37° water bath. One tube should be incubated for 2 minutes; the other tube should be incubated for 5 minutes.
Keep these two digestions separate in subsequent steps, until fractions are chosen for combining (Step 43).

26. Add 20 µl of MNase stop solution to each tube.

27. Chill the samples on ice.

Recovery of Soluble Chromatin Fractions (16 h)

28. Centrifuge the 1.5-ml tubes with the MNase-digested nuclei (from Step 27) to pellet the nuclei (10,000 rpm, 10 min, 4°C).

29. Transfer the supernatant into another 1.5-ml tube. Store it for up to 1 day at 4°C.
This supernatant contains the first soluble fraction of chromatin, S1, which comprises small fragments only. Do not discard the pellet.

30. Carefully resuspend the pellet in 500 µl of dialysis-lysis buffer.
At this stage, we normally proceed to Step 31; however, a more expedient lysis/dialysis procedure used in our laboratory has yielded chromatin fragments of comparable quality. It replaces Steps 31-34 as follows:

i. After resuspending the pellet in 500 µl of dialysis-lysis buffer, place the samples for 1 hour at 4°C.

ii. Centrifuge the samples (10,000 rpm, 10 min, 4°C) in a microcentrifuge.

iii. Proceed with Steps 35-36.

31. Close one side of the washed dialysis tubing (from Step 22) with a universal closure clamp. Transfer the 500 µl of resuspended nuclei (from Step 30) into the dialysis tube, and close the second side with another clamp.

32. Submerge the tube in 1-2 liters of dialysis-lysis buffer. Perform dialysis for 12-16 hours at 4°C with constant mild stirring using a magnetic stirrer.

33. Transfer the dialyzed nuclei into a 1.5-ml microcentrifuge tube.

34. Centrifuge the nuclei (10,000 rpm, 10 min, 4°C) in a microcentrifuge.

35. Transfer the supernatant in a new 1.5-ml microcentrifuge tube. Store it for up to 1 day at 4°C.
This is the second soluble chromatin fraction, S2, comprising the larger fragments of chromatin that were removed from the nuclei during lysis/dialysis (Step 32).

36. Resuspend the pellet in 50 µl of dialysis-lysis buffer. Store it for up to 1 day at 4°C.
This is chromatin fraction P.

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