HT-ChIP protocol –Amit lab July 2012
A High-Throughput Chromatin Immunoprecipitation Approach Reveals Principles of
Dynamic Gene Regulation In Mammals / Garber M. et al
ChIP module
Note: sonication and lysis conditions were calibrated to working with BMDCs. For optimal
ChIP in different cell types we suggest optimizing different lysis/sonication conditions.
Day 1:
Start with a 10cm petri dish, 10^7 cells in 10ml growth media per dish.
Stimulate BMDC cells.
1. Crosslink by adding 1% final formaldehyde concentration (use fresh formaldehyde ampulla
in every experiment): Put plates on the rotary shaker and start shaking slowly. Add 667ul of
16% formaldehyde per 10ml culture while shaking. Incubate for exactly 10min.
2. Add 520ul of 2.5M glycine (final concentration: 125mM) to each plate and continue shaking
for additional 5 min.
Glycine acts as a quencher. It is highly recommended to make fresh glycine every month
as the solution tends to change pH over time. At correct pH, medium color will change to
yellow.
3. Scrape cells and collect everything into 15 or 50ml tubes depending on the volume (do
everything on ice).
4. Spin at 1250 rpm for 5 min at 4C. Remove supernatant. Wash with 10ml of cold PBS and
spin again.
5. Remove supernatant. Transfer the pellet into 1.5 ml tubes and wash two more times.
At this stage crosslinked cells can be flash frozen at -80.
6. Cell lysis: Add 1ml RIPA+ protease inhibitors (PI) buffer for every 7.5x107 cells. Allow cells
to lyse for 10 min at 4C.
At this stage couple antibody to protein G magnetic beads:



Wash twice 75ul beads per sample with 300ul binding/ blocking buffer.
Incubate washed beads with the antibody (10ug for TFs antibodies, 3ul for
chromatin markers antibodies) in ~300 ul of binding/ blocking buffer, ~1hr, RT.
Wash beads with 300ul blocking/binding buffer.
7. Sonicate cells using Branson Sonifier with a cooling system according to calibrated
conditions (for BMDCs: 40% amplitude for 5.20 min: 1.3sec ON, 2sec OFF. Cooling system
should be at -4C).
1
8. After sonication spin for 10 min, max speed, at 4C. Transfer to a new 2 ml tube. Bring
volume to ~400ul lysate per IP (add RIPA+PI if necessary).
9. Take out 30ul of each sample and freeze at -20c till the next day – this will be used as
the input for each sample. Aliquot the remaining sonicated material to Protein G beads
coupled to the specific Ab. Tumble overnight at 4C.
Day 2:
10. Place the tube on the 1.5ml tubes magnet and remove supernatant. Add 180ul cold
RIPA+PI and transfer to a chilled 96 well plate.
11. Wash beads with 180ul cold RIPA buffer using a multi-channel pipette (picture #1). Do not
pipette beads, but simply move the plate on the magnet one row to the right (picture #2),
and then back to the left (picture #3). This will make the beads move from one side of the
well to the other side and back, and will result on good washing of the beads. Place plate
on the magnet for additional 30 seconds (picture #4) and aspirate supernatant (picture
#5).
12. Repeat washing with RIPA buffer five times in the same manner.
Picture #1: adding 180ul of RIPA buffer to each well, using a multi-channel pipette:
Picture #2: moving the plate on the magnet one row to the right:
2
Picture #3: moving the plate back to the left:
Picture #4: the beads attach to the magnet after a few seconds:
Picture #5: aspirating the supernatant using a multi-channel vacuum aspirator:
Continue washing in the same manner with additional buffers:
a) Wash twice with 180ul cold RIPA-500 wash buffer.
b) Wash twice with 180ul cold LiCl wash buffer.
c) Wash once with 180ul cold TE wash buffer.
3
13. After washing keep sample at RT and add 50ul of direct elution buffer. Add a sample of
WCE (from day 1 step 9) in this step for the remaining of the process: add 10ul WCE
+ 40ul direct elution buffer.
14. Add 2ul of RNase (Roche) + 3ul direct elution buffer, incubate at 37C for 30 min.
15. Add 1ul glycogen + 2.5ul Proteinase K(Invitrogen) + 1.5ul direct elution buffer. Incubate at
37C for 2hrs.
16. Reverse cross-linking: Incubate sample at 65C overnight.
After reverse cross-linking, continue to HT-ChIP-Seq library construction module, or
freeze samples at -20
.
Library construction module
Important comment: the first step (first SPRI cleanup) is described in details, including
pictures for clarification. All following SPRI cleanup steps should be done very similarly, except
for changes in buffers volume etc. The same protocol is used for automated liquid handling
robots like the Agilent Bravo.
1. First SPRI cleanup – use ChIP samples after reverse cross-linking. Sample volume at this
step should be 60ul.
 Add 140ul (2.3X) SPRI beads [Agencourt AmPure beads]. Carefully mix by pipetting 15
times (Pictures #1 and #2). After 15x mixing the sample should look homogenous
(picture #4). Incubate for 4 min at room temp.
Picture #1: mixing SPRI beads with ChIP samples:
4
Picture #2: mixing beads with samples – continue:
Picture #3: samples after 15x mixing look homogenous:
 Separate supernatant from beads by placing plate on the magnet for 4 min (pictures
#4-6).
Picture #4: placing plate on the magnet (beads are still in suspension):
Picture #5: plate on the magnet after 4 min (beads adhere to the side of the well)
5
Pictuer #6: plate on the magnet after 4 min, look from the top:
 Discard supernatant (vacuum aspiration is suggested, as shown in picture #7).
Picture #7: discarding supernatant by vacuum aspiration:
 Leave plate on magnet and wash beads twice with 100ul of 70% EtOH (picture #8) -
Incubate for 30 sec. without disturbing the beads, then discard supernatant.
Picture #8: Ethanol washing of the beads, while the plate is still on the magnet:
6





Move Eppendorf plate off magnet and air dry SPRI beads for 4 min, RT.
Elute in 60ul Tris-HCl pH 8.0. Mix by pipetting 15 times and incubate for 2 min, RT.
Place plate on magnet and incubate for 4 min.
Transfer 20ul of supernatant for backup into a new plate.
Continue with the remaining 40ul (including the beads) to end-repair step.
2. End Repair:
Important: Use pre-made buffer aliquots for 24 samples and discard leftover buffer after
single use avoiding repeated freeze-thaw cycles.
 Prepare mastermix in a 2 ml tube according to your sample number.
 Add 27ul to each well.
Reagent
final conc
μl/1rxn
48 samples (55rxn)
End-Repair Mix Buffer
1x
25
1375
T4 PNK (10U/ul)
0.15 U/ul
1
55
T4 POL (3 U/ ul)
0.04 U/ul
1
55
27
1485
Mix Total Volume
 Use the following program on a 96 well thermal cycler: 12°C for 15 min, 25°C for 15
min, 4°C hold.
3. End Repair SPRI clean up:
 Add 147ul of SPRI buffer (20% PEG NaCl 2.5M) to each well
 Pipette mix 15x and incubate for 2 min
 Separate supernatant from beads by placing plate on the magnet for 4 min; discard
supernatant
 Leave plate on magnet and wash beads twice with 100uL of 70% Ethanol w/o disturbing
the beads; discard supernatant.
 Move Eppendorf plate off magnet & air dry SPRI beads for 4 min at RT.
 Add 40uL of Tris-HCl pH 8.0 to each well
 Pipette mix 25x and proceed to A-Base Addition
7
4. A-Base Addition:
Important: Use pre-made buffer aliquots for 24 samples and discard leftover buffer after
single use avoiding repeated freeze-thaw cycles.
 Prepare mastermix in a 1.5 ml tube according to your sample number.
 Add 20 ul to each well
Reagent
final conc
μl/1rxn
48 samples (55rxn)
dA Mix Buffer
1x
17
935
Klenow exo (5 U/ul)
0.25 U/ul
3
165
20
1100
Mix Total Volume
 Use the following program on a 96 well thermal cycler: 37°C for 30 min.
5. A-Base Addition SPRI cleanup:
 132uL of SPRI (20% PEG NaCl 2.5M) buffer is added to each well
 Pipette mix 15x and incubate for 2 min
 Separate supernatant from beads by placing plate on the magnet for 4 min; discard
supernatant
 Leave plate on magnet and wash beads twice with 100uL of 70% Ethanol w/o disturbing
the beads; discard supernatant
 Move Eppendorf plate off magnet & air dry SPRI beads for 4 min at RT
 Add 21ul of Tris-HCl pH 8.0 to each well
 Pipette mix 25x and incubate 2 min at RT
 Place plate on magnet for 2 min and pipette 19ul supernatant into a new well and
proceed to Adaptor Ligation
6. Adaptor ligation:
 Prepare mastermix in a 2 ml tube according to your sample number.
 Add 34 ul to each well
1X
48 samples (55rxn)
8
Adaptor Ligation Mix
29
1595
DNA quick ligase
5
275
Important: Spin down Adaptor Plate before removing the cover to prevent cross
contamination of adaptors.
 Add 5 ul of Indexed oligo adaptor (0.75 uM) to each well.
 Use the following program on a 96 well thermal cycler: 25°C for 15 min.
7. Adaptor ligation SPRI cleanup:










add 58ul Tris-HCl pH 8.0
add 56 ul AmpPurBeads (SPRI beads)
Pipette mix 15x and incubate for 2 min
Separate supernatant from beads by placing Eppendorf plate on the magnet for 4 min;
discard supernatant
Leave plate on magnet and wash beads twice with 100uL of 70% Ethanol w/o disturbing
the beads; discard supernatant
Move Eppendorf plate off magnet & air dry SPRI beads for 4 min at RT.
Add 42ul of Tris-HCl pH 8.0 to each well
Pipette mix 15x and incubate for 2 min
Separate supernatant from beads by placing plate on the magnet for 3 min, transfer
40ul of supernatant to a fresh well
At this step samples can be kept in -20 degrees O.N
8. Enrichment:
 Prepare mastermix in a 1.5 ml tube according to your sample number.
 Add 10 ul to each well
Final conc.
1x
48 samples (55 rxn)
FWD+RVS Enrichment Primer
2
110
0.4
22
10x Pfu Ultra Buffer (stored @
4C!)
5
275
Pfu Ultra II Fusion
1
55
Nuclease Free Water
1.6
88
dNTP mix (25mM each)
10 mM
 Use the following program on a 96 well thermal cycler:
- 95C, 2 minutes
- 14 cycles: 95C 30 sec, 55C 30sec, 72C 60sec.
- 72C 10 min.
- 4C
9
9. Enrichment SPRI cleanup:
 Add 35ul of SPRI beads to the sample
 Pipette mix 15x and incubate for 2 min
 Separate supernatant from beads by placing plate on the magnet for 4 min; discard
supernatant
 Leave plate on magnet and wash beads twice with 100ul of 70% Ethanol w/o disturbing
the beads; discard supernatant
 Move 96 well plate off magnet & air dry SPRI beads for 4 min at RT
 Add 40μl of Tris-HCl pH 8.0 to each well
 Separate supernatant from beads by placing plate on the magnet for 3 min, transfer
supernatant (ChIP-Library) to a new plate.
 Pool samples and send pool for sequencing.
10
How to make Reagents
ChIP module:
1xTE:
Stock
Final
For 100ml
For 250ml
Tris-HCl pH 8.0
1M
10mM
1ml of 100xTE
2.5ml of 100xTE
EDTA pH 8.0
100mM
1mM
99ml
247.5ml
H2O
5% Na-deoxycholate (DOC):
Na-Deoxycholate
Stock
Final
For 100ml
For 250ml
powder
5% (weight /volume)
5gr
12.5gr
Up to 100ml
Up to 250ml
H2O
**2.5M Glycine:
Glycine
Stock
Final
For 100ml
For 250ml
powder
2.5M
18.76gr
46.9gr
100ml (final)
250ml (final)
H2O
**Heat to 65c, Filter
RIPA buffer:
Stock
Final
For 100ml
For 250ml
Tris-HCl, pH 8.0
1M
10mM
1ml of 100xTE
2.5ml of 100xTE
EDTA, pH 8.0
100mM
1mM
NaCl
5M
140mM
2.8
7ml
Triton x-100
10%
1%
10ml
25ml
SDS
10%
0.1%
1ml
2.5ml
DOC
5%
0.1%
2ml
5ml
83.2ml
208
H2O
11
RIPA-500:
Stock
Final
For 100ml
For 250ml
Tris-HCl, pH 8.0
1M
10mM
1ml of 100xTE
2.5ml of 100xTE
EDTA, pH 8.0
100mM
1mM
NaCl
5M
500mM
10ml
25ml
Triton x-100
10%
1%
10ml
25ml
SDS
10%
0.1%
1ml
2.5ml
DOC
5%
0.1%
2ml
5ml
76ml
190ml
H2O
LiCl wash buffer:
Stock
Final
For 100ml
For 250ml
Tris-HCl, pH 8.0
1M
10mM
1ml of 100xTE
2.5ml of 100xTE
EDTA, pH 8.0
100mM
1mM
LiCl
8M
250mM
3.125ml
7.81ml
NP-40
100%
0.5%
0.5ml
1.25ml
DOC
5%
0.5%
10ml
25ml
85.37ml
213.4ml
H2O
Direct elution buffer:
Stock
Final
For 100ml
For 250ml
Tris-HCl, pH 8.0
1M
10mM
1ml of 100xTE
2.5ml of 100xTE
EDTA, pH 8.0
0.5M
5mM
0.8ml
2ml
NaCl
5M
300mM
6ml
15ml
SDS
10%
0.5%
5ml
12.5ml
87.2ml
218ml
H2O
12
Binding / blocking buffer:
Stock
Final
For 100ml
For 250ml
BSA
powder
0.5%
0.5gr
2.5gr
Tween
100%
0.5%
0.5ml
2.5ml
Up to 100ml
Up to 250ml
PBS
Chromatin Immunoprecipitation Reagents:
16% Formaldehyde: Pierce PIR-28908
Protease Inhibitors: Roche 04-693-124
RNase, DNase free 500ug/1ml, Roche #11119915001
Proteinase K: Invitrogen #25530-049
Glycogen: Roche 10901393001 (20mg)
Protein G magnetic beads: Invitrogen cat#100.04D
All the other chemicals are from Merck (molecular biology grade chemicals).
13
Library construction module:
SPRI buffer (20% PEG, 2.5M NaCl):








146.1gr NaCl
200gr PEG8,000
Add H2O (molecular biology grade) up to 900ml
Stir on a magnetic stirrer until solids dissolve
Adjust pH to 5.5 with concentrated HCl.
Add H2O to final volume of 1lit.
Filter with a 0.45um filter bottle.
Store at room temperature.
End Repair: Buffer w/o enzymes: ER Mix
Reagent
final conc
μl/1rxn
48 samples (55rxn)
10X T4 DNA Ligase Buffer
(contains 10mM ATP)
1x
6.7
368.5
BSA (10mg/ml)
0.1 mg/ml
0.67
36.9
dNTPs (10mM)
0.1 mM
0.67
36.9
16.96
932.8
Nuclease free water
T4 PNK (10U/ul)
0.15 U/ul
1
55
T4 POL (3 U/ ul)
0.04 U/ul
1
55
27
1485
Mix Total Volume
A-Base Addition: Buffer w/o enzymes: +dA Mix
Reagent
NEB
buffer 2
dATP, 100mM
final conc
1x
10 mM
Nuclease free water
Klenow exo (5 U/ul)
Mix Total Volume
14
0.25 U/ul
μl/1rxn
48 samples (55rxn)
6
330
0.1
5.5
10.9
599.5
3
165
20
1060
Adaptor ligation: Buffer w/o enzymes: AL Mix
1X
48 samples (55rxn)
2x DNA Quick Ligase Buffer
29
1595
DNA ligase
5
275
Mix Total Volume
Enrichment: Use commercial Pfu buffer according to the Pfu enzyme used (NEB, Agilent etc.):
Final conc.
FWD+RVS Enrichment Primer
1x
48 samples (55 rxn)
2
110
dNTP mix (25mM each)
10 mM
0.4
22
10x Pfu Ultra Buffer (stored @ 4C)
1x
5
275
Pfu Ultra II Fusion
1
55
Nuclease Free Water
1.6
88
15
Reagents:
Item
Agencourt AMPure XP (SPRI beads)
Tris-HCl, 1M, pH8.0
Cat#
Company
T2694-1L
Beckman Coulter
Sigma
PEG 8000
NaCl
T4 PNK (10U/ul)
P5413-500G
T4 POL (3 U/ ul)
End-it-mix
10X T4 DNA Ligase Buffer
BSA (10mg/ml)
ATP(10mM)
dNTPs (10mM)
Klenow exo (5 U/ul)
NEB-M0203B-96
NEB buffer2
dATP, 100mM
Quick ligase
2X Quick Ligation buffer
96 barcoded adaptors
Pfu Ultra II Fusion
10x Pfu Ultra Buffer (@ 4C!)
dNTP mix (25mM each)
FWD+RVS Enrichment Primer
16
NEB-0201B-96
NEB-M0212B-96
NEB-B7002B-BB
Sigma
Sigma
NEB
NEB
Epicenter
NEB
NEB
NEB
NEB
NEB
NEB
NEB
NEB-M2200B-60
NEB-M2200-35
NEB
NEB
Broad-IDT
Agilent
Agilent
NEB
Broad-IDT