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Micrococcal nuclease preparation of native mononucleosomes
This protocol is a hybrid of the most effective parts of a few protocols and
represents the sum of eight side-by-side preparations to optimize each step. A
“Stillman” detergent lysis1, followed by a an “Umlauf/Turner2,3” Sucrose cushion
purification of the nuclei, then MNase digestion optimized to produce largely
mononucleosomes followed by sucrose gradient purification of
mononucleosomes (adapted from Turner/Allis lab)2,4.
Required buffers, reagents and materials
Buffer A (sterile filter)
10 mM HEPES pH 7.9
10 mM KCl
1.5 mM MgCl2
340 mM sucrose
10% (v/v) glycerol
Supplement to 50 µg/ml BSA
from NEB,
0.5 mM PMSF, 0.5mM
benzamidine,
5mM 2-mercaptoethanol, 1x
Roche protease inhibitor
cocktail
Sucrose Cushion
(sterile filter)
10 mM HEPES pH 7.9
30% (w/v) sucrose
1.5mM MgCl2
1M CaCl 2
100mM EGTA (pH ~8.9)
QIAquick PCR cleanup
kit
Worthington
Micrococcal Nuclease
25U/µl in milliQ water with
0.1% NEB BSA)—frozen
aliquots. NB: the unit
defintion of this enzyme is
far different than that of
the sigma enzyme (1 U
sigma ~ 15-25 U of the
Worthington enzyme).
5% Sucrose Buffer
10mM NaCacodylate, pH 7
(this is antimicrobial)
1mM EDTA
0.5mM EGTA
50 mM KCl
5% (w/v) sucrose
Supplement to 50 µg/ml
BSA from NEB, 0.5 mM
PMSF, 0.5mM benzamidine,
5mM 2-mercaptoethanol,
1x Roche protease inhibitor
cocktail
29% Sucrose Buffer
Same as 5% but with
29% (w/v) sucrose
instead.
Harvest Cells
This protocol works well for approximately 1x107 to 1x108 cells (between 4 and
35 15cm plates of near confluence HEK293 cells), and may very well work for
smaller and larger cell masses. Harvest cells by scraping and wash 2x in PBS, in
the second final wash, supplement the PBS with protease inhibitors and other
appropriate inhibitors (e.g., phosphatase inhibitors, HDAC inhibitors, etc.). Spin
down at 250 x g, 5’, 4°C and remove the supernatant. Assess the packed cell
pellet volume (PCV) and note it for use in this prep. The pellet may be flash
frozen at this point and stored indefinitely @ -80°.
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Detergent lysis and crude nuclei harvest
Resuspend the cells in 2.5 PCV of buffer A supplemented to 5 mM βME and with
protease inhibitors (and other appropriate inhibitors, for HDACs, I use 5 µl of a 1
mg/ml TSA stock in DMSO per 10 ml buffer (0.5 µg/ml) chilled on ice. Prepare
a similarly supplemented buffer A volume (2.5 PCV) with 0.2% (v/v) triton X100 detergent (20 µl detergent per 10 ml of buffer), and when the pellet is
completely homogenized (by pipetting and light vortexing), add this detergent
containing buffer A to the cell suspension, mix with a few inversions and allow to
stand for 10 minutes with occasional gentle mixing on ice.
After 10 minutes of cell lysis by detergent (effectively 0.1% v/v triton X-100),
pellet nuclei via centrifugation (1300x g, 5’, 4°C). The supernatant contains
soluble cystosolic proteins and some nuclear protein as well, and may be further
clarified with a high-speed spin to remove partially soluble debris1,5. In general
the detergent lysis of the Stillman fractionation releases more soluble nuclear
proteins than a Dignam-Roeder hypotonic/mechanical lysis1,5.
Clean-up crude nuclei with a sucrose cushion
Clean intact nuclei are the key to consistent and high yield MNase prepared
mononucleosomes. Cellular debris and inhomogeneity during the MNase digest
will severely impact reproducibility. In particular, lysed nuclei will severely impact
the consistency of the nuclease fragmentation by forming a viscous snot-like
mass that resists permeation by the nuclease, yielding over-digested material on
the surface and undigested material on the interior.
Resuspend the pellet in 6 PCV of buffer A (supplemented as before) by gentle
pipetting and inversion—homogeneity is critical here. Spin through a chilled 7.5
ml sucrose cushion in a 50 ml conical tube supplemented as with Buffer A (1300
x g, 12’, 4°C, swinging bucket tabletop). Note that gentle application to the top
of the cushion allows some mixing to occur at the interface—if you are too
careful though, the surface tension will prevent the nuclei from passing through
it. If the interface is still apparent, break the surface tension with gentle
agitation with a stripette. If some nuclei remain in the form of clumps atop the
cushion after the spin, harvest and mix the upper layers without disturbing or
contaminating the pellet, then reapply the homogenized layers to another
sucrose cushion and spin again.
In nucleo MNase digest and nucleosome recovery
On ice, gently resuspend the pelleted nuclei in 2 PCV of buffer A, supplemented
as before, this suspension should shimmer with a pearlescent luster of dilute
Pantene ProV shampoo. Measuring the nucleic acid concentration of this
suspension by UV spectroscopy is easier and more accurate than counting
nuclei. To accurately measure this, in triplicate, add 2µl of the suspension to 100
µl of 2M NaCl, bath sonicate for 30s and vortex vigorously. Measure the average
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A260 on the nanodrop and back calculate the DNA concentration in the nuclei
suspension. Dilute the suspension to achieve a nucleic acid concentration
between 1.2 and 1.6 µg/µl in DNA, and add CaCl2 to a concentration of exactly 1
mM from a 1M stock solution. Aliquot the suspension into 2ml eppendorf tubes,
and equilibrate in a 37°C water bath for 5-10 minutes. I divide my nuclei
suspension into two sets of aliquots, and digest one set for 6 minutes and one
set for 9 minutes. Initiate the digestion and carefully time each digestion by
adding 1U Worthington MNase for every 70 µg of DNA—this works out to be
~1.7-2 µl of 25U/µl MNase per tube and mix with a P1000. Stop the digestion
by adding 26 µl of 100mM EGTA solution mixing with a P1000, and placing on
ice. Pellet nuclei and nuclear debris at maximum speed in a eppendorf centrifµge
at 4°C for 5 minutes and recover the supernatant.
Until the prep is robust in your hands, I recommend running a gel to verify the
extent of the digest. Both 1.2% agarose in .5xTBE or 5-6% .5xTBE acrylamide
Novex DNA retardation gels work well here. You can load 15-20 µl of your
sample directly (it has sufficient glycerol and sucrose) to see a shifted but
diffuse nucleosome population (intact nucleosomes do not stain as well as naked
DNA with ethidium bromide or Sybr dyes). Additionally, take 50 µg of
nucleosome solution (~30-40 µl) and apply in with 600 µl of PB to a Qiaquick
PCR cleanup column. Wash with PE and elute extracted nucleosomal DNA in 30
µl EB (10 mM Tris-Cl pH 8). Add DNA loading buffer and load the 15-20 µl of
the extracted DNA in the same gel with a marker as well.
The crude mononucleosomes can be used at this point to do pilot experiments—
quantify the DNA content again—something in the range of 300-500 ng/µl is
typical. It is preferable to have clean mononucleosomes in order to make claims
of coexistence, thus further purification is required. You should also be able to
quantify the recovery of DNA at this point—re-extraction of the pellet with a
300 mM NaCl buffer can recover a bit more of the genome, however, it is in
larger oligonucleosome fragments (almost certainly representing
heterochromatin).
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Sucrose gradient isolation of mononucleosomes
Prepare the sucrose gradients during the MNase preparation if possible and store
on ice until ready for use. Using a gradient mixer, pour a 5 – 29% sucrose
gradient of the appropriate size. For large preparations I pour two 33ml SW28
gradients (loading more than 2 mg of nucleosomes per tube will not give clean
separation). For smaller preparations, SW40 gradients (11ml will work well,
36000 rpm, 15-16 hours)—do not load more than 400 µg here. If you are
going to use the fractions directly in IP, do not add reducing agent to the
sucrose gradient-- antibodies that may be susceptible to reductive inactivation.
Dilute the nucleosomes supernatant 1:1 with 10 mM HEPES pH 7.5, 100 µg/ml
BSA, 50 mM KCl—this will make the osmolality approximately equivalent to the
5% sucrose gradient starting point. Concentrate this solution in a 10K M.W.C.O.
biomax concentrator until the volume is ~500 µl per gradient tube. Carefully
apply the concentrated nucleosomes to the top of the gradient and spin for 2324 hours, 27000 rpm in SW28 rotor, 4°C. After centrifugation, fractionate with
cautious pipetting from the top of the meniscus into 30-40 fractions. Quantify
the A260 of each of these fractions (or a sampling of them) to get a sense of
where your mononucleosome peak resides. Take the fractions in this region and
Qiaquick extract the DNA as before, load onto a gel to see which fractions to
pool as a pure mononucleosome preparation.
The mononucleosome pool may then be dialyzed o/n against a suitable buffer
for IP, or a slightly lower salt buffer to maintain stability. I typically dialyze
against 50 mM HEPES pH 7.8, 50 mM KCl, 1mM EDTA in a snakeskin 7K M.W.C.O.
membrane. Further concentration with 100 µg/ml BSA, protease and other
inhibitors is usually required prior to IP.
References
1
Mendez, J. and Stillman, B., Chromatin association of human origin recognition complex,
cdc6, and minichromosome maintenance proteins during the cell cycle: assembly of
prereplication complexes in late mitosis. Mol Cell Biol 20 (22), 8602 (2000).
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2
3
4
5
O'Neill, L. P. and Turner, B. M., Immunoprecipitation of native chromatin: NChIP.
Methods 31 (1), 76 (2003).
Umlauf, in The Epigenome Network of Excellence.
Brand, M., Rampalli, S., Chaturvedi, C. P., and Dilworth, F. J., Analysis of epigenetic
modifications of chromatin at specific gene loci by native chromatin
immunoprecipitation of nucleosomes isolated using hydroxyapatite chromatography.
Nat Protoc 3 (3), 398 (2008).
Dignam, J. D., Lebovitz, R. M., and Roeder, R. G., Accurate transcription initiation by
RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids
Res 11 (5), 1475 (1983).
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