Digeston of HeLa Nuclei by Micrococcal Nuclease (MNase)
Posted by Erbay Yigit on Nov-11-2008.
Step 1. Nuclei Preparation:
- Starting material: ~100 µl HeLa cell pellet (~5-7x10^7 cells)
- Wash cells in ice-cold 1x PBS. Pellet at ~1,600 rpm 1 min
- Resuspend in ice-cold NP-40 lysis buffer
- Incubate on ice 5 min (not longer!)
- Pellet cells at 1,000 rpm, discard supernatant.
- Wash with 1 ml MNase digestion buffer.
- Resuspend cells in 1.5 ml of MNase digestion buffer.
Note: Make sure to add fresh proteinase inhibitors PMSF (0.5 mM f.c.) and benzamidine (BZA) (2 mM f.c.) into PBS, NP-40 lysis buffer, and MNase digestion buffers.
Step 2. Optimization of nuclei digestion to obtain mononucleosomal DNA:
If you are doing MNase digestion for the first time, there two important points you want to pay attention:
First, you want to know how much MNase you need to use for a given amount of nuclei for a successful digestion. Knowing this fact will make your life much easier, especially if you need to do MNase digestion repeatedly.
Second, how much DNA you need to run on an agarose gel to check the quality of your MNase digestion? If you do not load enough digested chromatin, you will not see it by ethidium bromide staining. On the other hand, if you load too much, you would be wasting your DNA and would not have nice resolution of nucleosomal bands.
Let's start with the first issue. The best way of addressing this problem is either to count the number of cells or to determine the weight the cell pellet. Either of these methods will give you a constant digestion pattern provided that your nuclei preps are similar in qualities. Alternatively, you can measure the amount of starting material by reading the UV absorbance of DNA. To do so follow the steps below.
Determine the amount of DNA/chromatin by UV spectrophotometer:
- Digest 0.3 ml nuclei (or enough amount) by MNase for about 10 min at 37 °C. It does not matter if you overdigest it.
- Stop the reaction by adding 1/10V (30 µl) of 250 mM EDTA.
- Add 0.9 ml 1M NaOH onto 0.1 ml digested nuclei to denature everything.
- Prepare blank in the same way, making sure that blank contains proteinase inhibitors. BZA gives absorbance at 260 nm. Therefore, you should not use absorbance at 260 nm to calculate DNA concentration. Instead, read the full spectrum from 220 to 800 nm if possible to see how graph looks, and determine the concentration of DNA based on the absorbance at 280 nm. DNA absorbance at 280 nm is half of the value at 260 nm. Therefore, you need calculate the concentration of DNA by follwoing formula. DNA concentration = (33 µg/ml) x (Abs at 280 nm) x (dilution factor) x 2.
The second issue is also easy to address. What you need to do is to load several hundreds of nano grams of digested chromatin on each agarose well. This amount is enough to see the digested DNA by ethidium bromide staining. If you know the concentration of DNA, just take enough material to load in the agarose gel. If you do not know the concentration of DNA, you need to know the number of the nuclei you used. Since the genome size is available for many model organisms, you can simply calculate how much DNA you would get from a single nuclei.
Test Digestion of Nuclei
- Add 0.3 ml (~20% of initial pellet) nuclei into a tube. Warm up nuclei at 37 °C for several minutes.
- Add 1 µl of MNase (50U/µl) into 300 µl nuclei. Immediately mix by pipetting and start timing.
- Remove 50 µl digested nuclei at 1 min, 2 min, 4 min, 8 min, 16 min, 32 min, etc. time points. Stop the reaction immediately by
adding 5 µl (1/10 of V) of 250mM EDTA. Then add 1/10V (5µl) of 10% SDS onto digested nuclei to break nuclear membrane.
- Bring the volume of nuclei up to 0.4 ml, which makes it easy to handle.
- Add 1/10 V (40µl) of 5 M NaCl (helps dissociate histones and other proteins from DNA).
- Extract with Phenol/Chloroform, and then with Chloroform only (1x Vol)
- Ethanol precipitation is not necessary here if you are careful enough not removing any organic phase.
- Remove RNA by RNAase Afor half an hour at 37°C
- Extract with Phenol/Chloroform, and then with Chloroform alone.
- Add 1/10 V of Na Acetate pH 5.2 and glycogen (optional) to precipitate. Note: If you are adding glycogen, you are risking of contaminating your DNA with the DNA which comes from the organism glycogen extracted. Please, consider this possiblity.
- Ethanol precipitate, resuspend in 20 µl of TE buffer. Load 10 µl onto agarose gel. Note: Avoid adding bromophenol blue and xylene cyanol dyes in DNA samples. You have to cut your DNA from gel you may contaminate them with dyes. Instead, I recommend plain glycerol or Orange G, which runs faster than almost any size of DNA.
Step 3. Agarose Gel Electrophoresis of Digested Chromatin
- Run in 3.3% NuSieve agarose (pour 100 mL agarose with thin-wide combs).
- Run in 0.5x Tris glycine running buffer without ethidium bromide.
- Runa at 80 V for until BB reaches bottom of the gel, which takes about 5 and half hour.
NP-40 lysis buffer:
10 mM Tris [pH 7.4], 10 mM NaCl, 3 mM MgCl2, 0.5% NP-40, 0.15 mM spermine, 0.5 mM spermidine (Enver et al, 1985).
MNase digestion buffer:
MNase stop buffer: 250 mM EDTA, 10% SDS.
5x Tris-Glycine running buffer:
0.25 M Tris, 2.0 M Glycine. There is no need to adjust the pH of this buffer. (Edgar Bonste and Peter B. Becker, Methods in Molecular Biology, Vol. 119:Chromatin Protocols).
PMSF (Phenylmethylsulfonyl fluoride) stock:
50 mM in Ethanol.
Benzamidine (BZA) stock:
MNase digestion buffer:
Tris [pH 7.4] 10 mM
NaCl 15 mM
KCl 60 mM
Spermine 0.15 mM
Spermidine 0.5 mM
CaCl2 1 mM
NP-40 lysis buffer for nuclei preparation from HeLa cells:
10 mM Tris [pH 7.4], 10.00 mM
10 mM NaCl 10.00 mM
3 mM MgCl2 3.00 mM
0.5% NP-40 0.50 %
0.15 mM spermine 0.15 mM
0.5 mM spermidine 0.50 mM