Chromatin Immunoprecipitation 2014

Chromatin Immunoprecipitation (ChIP)
Required Solutions
10x PBS
80 g NaCl
2 g KCl
17.4 g Na2HPO4.7H2O
2.4 g KH2PO4
Adjust pH to 7.4 and add H2O to 1 l
Lysis buffer
1% SDS
10 mM EDTA pH 8.0
50 mM Tris-HCl pH 8.0
add before use:
1 mM PMSF
1µg/ml leupeptin
1µg/ml Aprotinin
1µg/ml Pepstatin
or replace Leupeptin/Aprotinin/Pepstatin with Roche protease inhibitor tablets (cOmplete, EDTA-free
for 50 ml [#11697498001]; cOmplete mini, EDTA-free for 10 ml [#04693159001])
IP dilution buffer
0.01% SDS
1.1% Triton X-100
1.2 mM EDTA
16.7 mM Tris-HCl pH 8.0
150 mM NaCl
add before use:
1 mM PMSF
1µg/ml leupeptin
1µg/ml Aprotinin
1µg/ml Pepstatin
or replace Leupeptin/Aprotinin/Pepstatin with Roche protease inhibitor tablets (cOmplete, EDTA-free
for 50 ml [#11697498001]; cOmplete mini, EDTA-free for 10 ml [#04693159001])
IP Wash A
0.1% SDS
1% Triton X-100
2 mM EDTA pH 8.0
20 mM Tris-HCl pH 8.0
150 mM NaCl
add before use:
1 mM PMSF
1µg/ml leupeptin
1µg/ml Aprotinin
1µg/ml Pepstatin
or replace Leupeptin/Aprotinin/Pepstatin with Roche protease inhibitor tablets (cOmplete, EDTA-free
for 50 ml [#11697498001]; cOmplete mini, EDTA-free for 10 ml [#04693159001])
IP Wash B
0.1% SDS
1% Triton X-100
2 mM EDTA pH 8.0
20 mM Tris-HCl pH 8.0
500 mM NaCl
IP Wash C
0.25 M LiCl
1% NP-40
1% Na-Deoxycholate
1 mM EDTA pH 8.0
10 mM Tris-HCl pH 8.0
TE (4th IP wash)
10 mM Tris-HCl pH 8.0
1 mM EDTA pH 8.0
SDS-Na Carbonate
1% SDS
0.1 M NaHCO3
Make fresh from 10% SDS and 1 M NaHCO3
Denaturating solution
1.5 M NaCl
0.5 M NaOH
Neutralizing solution
3 M NaCl
0.5 M Tris-HCl pH 7.0
1 M NaPi pH 7.2
In 2.3 liters (orange capped roller bottle filled to shoulder).
308 g Na2HPO4
9.2 ml H3PO4
add ddH2O to close to final volume and adjust pH to 7.2 with H3PO4
adjust volume to 2.3 l
Church Mix
500 ml 1 M NaPi pH 7.2 (see below)
2 ml 0.5 M EDTA pH 8.0
70 g SDS
10 g BSA
ddH2O to 1 l
Heat to 55°C to dissolve. Filter through a 0.45 µm filter.
10x OLB (10x oligo nucleotide labeling buffer)
0.5 M Tris-HCl pH 6.8
0.1 M MgOAc
1 mM DTT
0.5 mg/ml BSA
for 1 ml of 10x OLB add 6 µl of 100 mM dGTP, dATP, TTP
make small aliquots (nucleotides are unstable) and store at -80°C, avoid freeze and thaw
ChIP-Grade Protein G Magnetic Beads (Cell signaling, #9006)
Ready to use
Hybond-N membrane (GE Healthcare)
DNA templates for labeling reaction
• Telomeric probe: 800 bp [TTAGGG]n Sty11 insert, (~20 ng/µl)
Isolate fragment from pSP73.Sty11x #21 digested with EcoRI on an agarose gel, use [CCCTAA]3
oligo (1ng/µl) for labeling reaction
• BamHI repeat probe for mouse DNA: 1 kb insert, (~20 ng/µl)
Isolate 1 kb fragment from plasmid #7320 (pBLUE KS- backbone) digested with EcoRV on an
agarose gel, use random hexamer primers for labeling reaction (Applied Biosystems, S06405, 50
µM)
• Alu repeat probe for human DNA: ~ 2 kb insert, (~20 ng/µl)
Isolate ~ 2 kb fragment from plasmid #53 (pBLUE KS- backbone) digested with XbaI and HindIII
on an agarose gel, use random hexamer primers for labeling reaction (Applied Biosystems,
S06405, 50 µM)
ChIP timeline:
Day 1: Prepare lysate and do the IP o/n
Day 2: wash the IPs, reverse crosslinks and extract the DNA, do precipitation o/n or 2 hours
Day 3: resuspend DNA, dot blot and hybridize o/n or for 6 hours
Day 4: Wash the membrane and expose
*Day 2 and 3 can be combined.
Preparing the lysate
1.
Grow cells to subconfluence. Typically, harvest 1-2 ∅ 15 cm dishes per condition.
2.
Crosslinking:
a. Crosslinking done on cells on plate
i. Remove medium and add 15 ml PBS/1% Formaldehyde (from 37% solution).
ii. Swirl on platform at RT 10-30 min
iii. Add 2 ml 1.5 M Glycine (final concentration 0.2 M) to stop crosslinking
iv. Swirl at RT for 5 min
v. Wash 2 times with cold PBS
vi. Scrape cells in ~ 10 ml PBS into 50 ml conical tube. Spin down cells. (for the
estimation of cell numbers, count cells on a plate harvested in parallel by
trypsinization)
b. Crosslinking done on trypsinized cells
i. Harvest cells by trypsinization; collect in a 50 ml tube, add sufficient medium with
serum to inactivate the trypsin (15 ml medium/ 1.5 ml trypsin and count cells),
count cells and spin down
ii. Add 15 ml PBS/1% Formaldehyde (from 37% solution).
iii. Crosslink at RT for 10-30 min on a nutator
iv. Add 2 ml 1.5 M Glycine (final concentration 0.2 M) to stop crosslinking
v. Mix on a nutator for 5 min
vi. Wash 2 times with cold PBS, spin down cells in between the washes.
3.
Combine cell pellets in cold PBS with 1 mM PMSF. Spin again. Place cell pellet on ice or store
at -80°C.
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4.
Resuspend pellet in lysis buffer + protease inhibitors to a concentration of 2x10 cells/ml. The
buffer needs to be at RT, but keep the lysate on ice from this point on. The lysate can be more
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concentrated if necessary, but no more than 5x10 cells/ml.
5.
Incubate for 15 min on ice.
6.
Sonicate in ice/water using the Bioruptor (standard conditions: 1.5 ml tubes, 100-300 µl per tube,
power setting H (high), sonication cycle 30 sec ON, 30 sec OFF, sonication time 10 min. If other
tubes are used, choose the right adapters and optimize sonication conditions.) The lysate
should clear up. Check between cycles to make sure that the lysate remains ice-cold. Optional:
Check for sonication on a gel. To do this, take an aliquot before and after sonication, reverse
crosslinks and extract the DNA as outlined below. Bulk DNA should be sheared to 0.8-1 kb.
7.
After sonication, spin down at max speed for 10 min at RT (pool supernatants if necessary).
Immunoprecipitations
1.
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6
Use 100-200 µl lysate (1x10 - 2x10 cells [to immunoprecipitate shelterin proteins in MEFs
500’000 cells are enough]) per IP. On ice, add 1 ml of IP dilution buffer + protease inhibitors
(fresh). Mix by gently inverting the tubes a few times, let sit on ice for 10 min, and add the
antibody. Incubate o/n at 4°C on a rotator.
For our rabbit polyclonal sera, use crude serum and include the pre-immune controls. Usually 15
µl – 20 µl of crude serum is good. 1-2 µg of purified antibodies can be used a reference amount.
Optimal conditions have to be tested.
aTRF1: human - #371, 20 µ; mouse - #1448/1449, 15µl
aTRF2: human - #647, 20 µl; mouse - #1254/1255, 15µl
aRAP1: human - #765, 20 µl; mouse - #1252/1253, 15µl
aTIN2: human - #864, 20 µl; mouse - #1446/1447, 15µl
aPOT1: human – Abcam Ab124784, 4 µl; mouse not available
aTPP1: human - #1151, 20 µl; mouse not available
2.
Remember: Keep one aliquot of 50 µl lysate (50% of amount used for immunoprecipitation) o/n
at 4°C, as ‘input’ fraction, to be processed the next day. Inputs will be processed with the other
samples after the immunoprecipitation step.
3.
The next day: Add 30 µl magnetic beads (Cell signaling, #9006) to each IP. Incubate another 30
min at 4°C on the rotator.
4.
Wash the beads once with 1 ml of each of the buffers: A, B, C, and TE. Add protease inhibitors
only to buffer A. Vortex and place on ice in between washes.
5.
Elute the immunoprecipitated material as follows: At the end of the last wash, re- pellet the
beads, remove as much buffer as possible, and resuspend beads with 250 µl of 1% SDS/0.1 M
NaHCO3 (freshly diluted) by vortexing. Incubate for 10 min at RT. Pellet the beads, transfer
supernatant to a fresh tube, and wash the beads again with 250 µl of SDS-carbonate. Incubate
at RT 10 for min, pellet beads, and pool supernatant with the previous eluate.
6.
Important: this is the step where the input samples start to get processed. For the input samples
bring the volume up to 500 µl with SDS-carbonate (i.e. add 450 µl to the 50 µl of lysate set
aside)
7.
To all the samples, add 20 µl of 5 M NaCl, mix, and incubate at 65°C for 4 to 6 hours to reverse
the crosslinks..
8.
After 4-6 hours at 65°C, add 10 µl of 0.5 M EDTA and 20 µl 1M Tris-HCl pH 6.5.
9.
Add 20 µg RNAse A (20 µl of 1 mg/ml, heat inactivated, DNAse-free), incubate at 37°C for 30
min.
10. Add 40 µg of Proteinase K (20 µl of 2 mg/ml freshly made stock). Incubate for 1 hour at 37°C.
11. Extract the samples with 0.5 ml phenol/chloroform/isoamylalcohol. Transfer the aqueous phase
to an Eppendorf tube and add 20 µg of glycogen (from 20 mg/ml stock) and mix. Add 1 ml 100%
of EtOH to precipitate the DNA. Precipitate o/n at -20°C. Note: In order to keep the volume low
and to do the precipitation step in 1.5 ml Eppendorf tubes (2 ml tubes don’t work well because of
their shape), we only add 2 volumes of EtOH instead of 2.5 volumes. This EtOH concentration is
suboptimal (66% instead of 75% EtOH).
12. Spin down for 20 min in microfuge at max speed at 4°C. Remove all EtOH after second spin
down. Resuspend pellet in 10 mM Tris-HCl pH 7.5.
Dot Blotting and hybridization
1.
For dot blotting, first prepare the manifold and membranes. Then boil DNA samples for 5 min,
snap cool on ice, quick spin in microfuge, and load.
2.
With the dot blot manifold, use two 3MM Whatman papers and Hybond-N membrane (11 cm x
7.5 cm), all saturated with 2x SSC (pre-wet membrane first by floating it on top of H2O, then soak
in 2x SSC). Assemble Whatman papers and membrane on the manifold and ensure that the
vacuum is working well by loading 2x SSC on a few wells. Then switch the vacuum off until you
are ready to load. Beware to not let the membrane dry out.
3.
For loading, pipet 200 µl of 2x SSC in each well. Then load the samples and mix with the 2x
SSC (alternatively mix 2x SSC and sample in Eppendorf tubes). This ensures an even
distribution of the sample on the membrane.
4.
Load 30-50 µl (80 µl for IMR90 cells) on dot blot to probe for telomeric DNA with the Sty11 insert,
load 10-20 µl on membrane to probe for Alu (human cells) or BamHI repeats (mouse cells).
When ready, switch on the vacuum. Optional: Load 1, 2, and 4 pg Sty11 plasmid to verify that
the range is linear in the quantitation. The total fractions usually fall in that range. For the input
samples it is a good idea to load two different dilutions.
5.
Denature the membrane by placing it on a 3MM Whatman saturated with denaturating solution.
Denature for 10 min.
6.
Neutralize the membrane by placing it on a 3MM Whatman saturated with neutralizing solution
for 10 min.
7.
Remove excess liquid by gently blotting on dry paper. Crosslink the DNA to the membrane
immediately in the Stratagene UV crosslinker, DNA side up (Auto crosslink program). Rinse in
2x SSC.
8.
Prehybridize in medium size Hybaid bottle with 7 ml Church buffer for 30 min at 65°C.
9.
Add filtered mixture of Church mix and probe (7 ml – for details on probe preparation see also
Telomere Blot protocol) and hybridize o/n at 65°C. 1/4 of the Sty11 probe prepared for 20 cm x
20 cm membranes is enough for the ChIP membrane hybridization.
• 2.5 µl DNA template [20ng/µl]
• 1.25 µl oligo
• 6 µl ddH2O
• Mix, boil 5 min, spin for 5 sec to get condensation down, cool on ice, and add:
• 1.25 µl 10xOLB with dATP, dGTP, dTTP
• 1.25 µl 32P-alpha-dCTP (3000 Ci/mmol)
• 0.25 µl Klenow polymerase
• Mix, incubate 90 min at RT (or longer), add ddH2O up to 50 µl and purify over a G-50
column, add to Church mix and filter trough 0.45 µm filter.
10.
Washes: wash the membrane in 2x SSC at RT for 5 min. Wash 2-3 times. If necessary, perform
the following washes, but carefully monitor the blot after each wash. Expose as soon as nonspecific counts appear to have disappeared (e.g. in the corners): 2x SSC/RT, 0.2x SSC/RT
(usually this is enough), 2x SSC 0.1%SDS/RT, 0.2x SSC 0.1% SDS/RT. Perform same
sequence of washes at 65°C if necessary.
11.
Expose on to a phosphorImager screen (usually a few hours is enough).
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