Day 1
GENERATION OF A HI-C LIBRARY FROM MAMMALIAN CELL CULTURE USING DPNII
1. CROSSLINKING OF CELLS
Grow the cells in appropriate culture medium. 5x 106 cells are enough for making one Hi-C library. It is very
important to estimate the number of cells accurately because it affects the complexity of the Hi-C library.
1.1)
1.2)
1.3)
1.4)
1.5)
1.6)
1.6.1)
1.6.2)
1.7)
1.1.1)
1.2)
1.3)
1.4)
1.5)
1.6)
1.7)
1.8)
1.9)
1.10)
1.11)
1A. FOR ADHERENT CELLS:
Aspirate the medium, wash with ~5 ml HBSS and add 22.5 ml of HBSS (no serum!) per plate.
Crosslink the cells by adding 625 µl of 37% formaldehyde to obtain 1% final concentration. Mix gently,
immediately after addition of formaldehyde.
Incubate at room temperature (RT) for exactly 10 min. Gently rock the plates every 2 min.
To quench the crosslinking reaction, add 1.25 ml of 2.5 M glycine, mix well
Incubate for 5 min at RT and then incubate on ice for at least 15 min to stop crosslinking completely.
Scrape the cells from the plates with a cell scraper and transfer to a 50 ml tube.
Spin down cells 800xg for 10 min
Wash with PBS
Cells can be snap-frozen in liquid nitrogen or dry ice for 20 mins and stored at -80°C for at least 1.5 years
or one can continue with cell lysis.
1B. FOR SUSPENSION CELLS:
Add 1.25 ml of 37% formaldehyde to 45 ml of HBSS or fresh culture medium or (without serum!)
in one shot and mix by inverting the tube several times.
Gently pellet the cells by spinning at 300xg for 10 min at RT.
Discard the supernatant.
Resuspend the pellet in 45 ml of fresh culture medium without serum and with formaldehyde
(prepared in step 1.1) Break cell clumps by pipetting up and down.
Incubate at room temperature (RT) for exactly 10 min. Gently invert the tube every 1-2 min.
To quench the formaldehyde in the reaction, add 2.5 ml of 2.5 M glycine in one shot, mix well.
Incubate for 5 min at RT and then incubate on ice for at least 15 min to stop crosslinking completely.
Split the crosslinked cell suspension into aliquots of 5x 106 cells.
Centrifuge the crosslinked cells at 800xg for 10 min.
Discard the supernatant by aspiration. It is important to remove the liquid phase completely.
Cells can be snap-frozen in liquid nitrogen or dry ice for 20mins and stored at -80°C for at least 1.5
years or one can continue with cell lysis
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Day 2
2. CELL LYSIS AND CHROMATIN DIGESTION (WITH DPNII)
NB!
This protocol is set up to generate libraries from ~ 5x106 of cross-linked cells. We have successfully
generated libraries for next generation sequencing from as little as 5x105cells. However, decreasing the
number of cells usually leads to an increase in PCR cycles and a subsequent reduction of library
complexity!
Make sure that for steps 2.1-2.15, the buffer used is paired to the restriction enzyme (i.e. NEBuffer 3.1 for DpnII
or NEBuffer 2.1 for HindIII)
2.1)
2.2)
2.3)
2.4)
2.5)
2.6)
Resuspend one crosslinked cell aliquot (~5x 106 cells) in 1 ml of ice-cold lysis buffer containing 10μl
protease inhibitor cocktail (100x, Thermo)
2.1.1) Incubate on ice for 15 min.
Dounce homogenize the cells on ice with pestle A.
2.2.1) Slowly move pestle up and down 30 times
2.2.2) Incubate on ice 1 min to let the cells cool down
2.2.3) Then do 30 more strokes.
Transfer the lysate to a 1.7ml tube
Centrifuge for 5 minutes at 2,500xg at RT.
Discard the supernatant and then wash the pellet twice by resuspending it in 500μl of ice cold 1x
NEBuffer 3.1 and then centrifuging the sample for 5 min at 2,500xg.
Resuspend the pellet in 1x NEBuffer 3.1, so that the total volume of the suspension is 360 µl.
(Optional) Save 18 µl of lysate for the CHROMATIN INTEGRITY CONTROL*:
Take 18 µl of lysed cells from the previous step. Add 50 μl of 1x NEB and 10 μl of Proteinase K (10
mg/ml). Incubate for 30 minutes at 65°C. Purify DNA by single phenol-chloroform extraction without
ethanol precipitation. Add 1μl of RNAseA (1 mg/ml) to the aqueous phase and incubate for 15 min at
37°C. Check quality of the sample by running it on 0.75% agarose gel. The sample is good if DNA is
either stuck in the well or runs as a single high molecular weight band (>23 kb)
After taking 18µl lysate control we have 342 µl remaining total volume.
2.7)
2.8)
2.9)
2.10)
2.11)
Add 38 μl of 1% SDS to each Hi-C tube (380 µl total). Mix carefully by pipetting up and down. Avoid
making bubbles (0.1% SDS final).
Incubate at 65°C for 10 minutes exactly to open chromatin
Place tubes on ice immediately after
Add 43 μl of 10% Triton X-100 to the Hi-C-tube (423 µl total) to quench the SDS (1% Triton final).
Mix gently by pipetting up and down Avoid making bubbles.
Add 400 Units of DpnII to each tube:
NB!
2.12)
2.13)
The addition of the following ingredients depends on the enzyme and its concentration.
Add 12 µl of 10x NEBuffer 3.1 to the Hi-C tube to compensate for added components (435 µl total
before DpnII)
Add 400U (40 µl of 10,000 units/ml) DpnII to the Hi-C tube (475µl total), mix gently Digest the
chromatin overnight at 37°C on a rocking platform or in a ThermoMixer.
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Day 3
3. M ARKING OF DNA ENDS WITH BIOTIN-14-DCTP
3.1)
Incubate tube at 65˚C for 20 mins in order to deactivate the endonuclease enzyme.
NB!
This inactivation depends on the enzyme used. Please check enzyme datasheet
(Optional) Take 10 µl and keep it aside TO ASSESS THE DIGESTION on gel. Replace lost volume with 10µl
of 1x NEBuffer 3.1
For the aliquots taken at steps 2.7 and 3.1, add up to 50 μl of appropriate 1x NEB and 10 μl of
Proteinase K (10 mg/ml).
3.1.1)
3.1.2)
3.1.3)
3.1.4)
Discard supernatant and proceed to ligation on beads
Incubate for 30 minutes at 65°C.
Purify DNA by single phenol-chloroform extraction without ethanol precipitation.
Add 1 μl of RNAseA (1 mg/ml) to the aqueous phase and incubate for 15 min at 37°C.
The sample quality can be assessed by running it on 0.75% agarose gel. DNA from step 2.7 is either stuck
in the well or runs as a single high molecular weight band (>23 kb), whereas the digested DNA from step
3.1 should run as a smear between ~200-3000 bp. Perform this control before the addition of biotin in
the next step.
3.2)
Prepare a Biotin Fill-in MasterMix as follows:
FILL-IN MIX (ADD IN THIS ORDER)
milliQ
10x NEBuffer 3.1*
10 mM dCTP
10 mM dGTP
10 mM dTTP
0.4 mM biotin-14-dATP**
5U/ μl DNA polymerase I Klenow
TOTAL
PER TUBE
2 µl
6 μl
1.5 μl
1.5 μl
1.5 μl
37.5 μl
10.0 μl
60 µL
*The same buffer that was used for digestion (NEBuffer 3.1 for DpnII or NeBuffer 2.1 for HindIII)
**The biotinylated dNTP is determined by on the overhang created by the endonuclease used for
digestion. For DpnII, generating a GATC overhang we use biotinylated-dATP instead of BiotinylateddCTP used for HindIII
3.3)
3.4)
3.5)
3.6)
Put the tubes on ice until cooled to room temperature:
Add 60 μl of the Fill-in master mix to each Hi-C tube (535 µl total)
Mix gently by pipetting up and down without producing any bubbles
Incubate the tubes at 23°C for 4 hours in a ThermoMixer (900 rpm mixing; 10 secs every 5 mins)
3.6.1) Prepare ligation mixture (below) during incubation
3.6.2) Place the tubes on ice immediately after incubation
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Day 3
4. BLUNT END LIGATION
4.1)
Prepare the 665 µl ligation mix during fill-in:
LIGATION MIX
Water
5x ligation buffer [Invitrogen]
10% Triton X-100
10 mg/ml BSA
T4 DNA ligase [Invitrogen]
TOTAL
4.2)
4.3)
PER LIBRARY
243μl
240 μl
120 μl
12 μl
50 μl
665 µL
N LIBRARIES
N* 665 µL
Add 665 µl of the ligation mix to each Hi-C tube (1,200 µl total)
Incubate at 16°C for 4 hours in ThermoMixer with interval shake
5. REVERSE CROSSLINKING
5.1)
5.2)
Add 50 μl of 10 mg/ml proteinase K to each tube and incubate at 65°C FOR AT LEAST 2 HOURS
Add another 50 μl of proteinase K to each tube and continue incubating at 65°C overnight (1,300 µl
total)
NB!
When running out of time, perform reversal overnight and add the second batch the next day
6. DNA PURIFICATION
6.1)
For each Hi-C tube, prepare 2x 15 ml tubes enabling 2 consecutive phenol:chloroform treatments
6.1.1) Mark 2x 15 ml PhaseLock tubes (see recipe/preparation) per library
6.1.2) Spin the tubes at ≥1500xg to move the grease to the bottom of the tube
6.1.3) Add 2.4 ml saturated phenol:chloroform (1:1; pH=8.0) to 2x 15 ml P tubes on top of each
pre-spun tube containing PhaseLock
NB!
6.2)
6.3)
Use appropriate gloves and labcoat when handling phenol:chloroform
Cool reaction mixtures after reverse crosslinking from 65˚C to RT
Extract the DNA:
6.3.1) Take 1.2 ml Hi-C sample the 15 ml containing 2.4 ml saturated phenol:chloroform and
PhaseLock (total ~3.6 ml)
6.3.2) Vortex for 30-60 seconds to obtain a homogenous, milky solution. Transfer the mixture to a
pre-marked 15 ml Phase Lock tube (heavy) to facilitate the phase separation step 6.1.2
6.3.3) Centrifuge at 3000xg for 5 minutes (room temperature)
Perform this extraction step twice by applying the liquid (top) phase to the next PhaseLock tube.
6.3.4)
6.3.5)
6.3.6)
6.3.7)
Transfer the aqueous phase with DNA (~1.2 ml) to a clean 15 ml tube (adequate for highspeed centrifugation at 16,000xg) and add 1 x TE (pH=8.0) up to 2 ml.
Add 1/10 volume of 3M sodium acetate, pH=5.2 per tube (120μl) and mix well.
Add 2.5 volumes of 100% ethanol (3 ml) and mix well by inverting the tubes several times.
Incubate the tubes on dry ice for ~20 min (or at -80˚C) until the sample becomes viscous but
not solid.
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Day 4
6.4)
6.5)
6.6)
6.7)
6.8)
6.9)
6.10)
6.3.8)
6.3.9)
Centrifuge tubes at 16,000xg for 30 min at 4°C.
Decant the supernatant with extra caution as the pellet detaches from the tube wall quite
easily and air dry the DNA pellets very briefly (~1 min).
NB!
The pellet might become invisible while drying out, so it is useful to mark the borders of the
pellet with a pen on the tube wall.
Dissolve the pellet on 450µl of 1x TLE (pH=8.0) and transfer to a 2 ml, 30kD Amicon Column.
Centrifuge at 14,000xg in tabletop centrifuge for ~5 minutes
Wash the column with 450 µl TLE and repeat this step twice
After the last wash, determine the volume on the column and adjust to ~ 100 µl for the Hi-C tube
Flip the column onto a new Amicon container tube
Spin at 14,000xg for 30 seconds to recover the DNA from the column
Add 1 µl of RNAseA (1 mg/ml) to each sample and incubate for 30 mins at 37°C (in Thermo mixer or
waterbath)
NB!
DNA samples can now be stored at -20°C.
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Day 4
7. QUALITY CONTROL OF HI-C LIBRARIES
7.1)
Run 2μl and 6 μl of a 1:10 dilution of each Hi-C and 3C library on a 0.8% agarose gel for quality
control
NB!
7.2)
7.3)
At this step the optional chromatin integrity control can be run alongside on the same gel
Quantify amount of DNA by densitometry. Use several dilutions of the 1kb DNA ladder (for instance
200, 100and 50 ng) as a standard curve to estimate the DNA concentration more accurately.
Measure the Biotin Incorporation by a PCR digest:
7.3.1) Perform a PCR to amplify one neighboring interaction in Hi-C as follows:
INGREDIENT
1 REACTION
Template DNA
800 ng
10X PCR buffer
10 µl
50mM MgSO4
8.0 µl
25mM dNTP
1.0 µl
primer_1 (10 µM)
4 µl
primer_2 (10 µM)
4 µl
Taq DNA polymerase
1 µl
MILLIQ
TO 100 µL
Step
1
2
3
4
5
6
7
8
Temperature
95°C
95°C
65°C
72°C
Go to 2
95°C
65°C
72°C
Time
5 minutes
30 seconds
30 seconds
30 seconds
34 times
30 seconds
30 seconds
8 minutes
One might use the following pairs of primers:
Primer PairA
Human
hDpnII-01
ENr313_DpnII_Anchor2
CTACAAGCAAGCTCAGAAGGTGATTCTTCA
hDpnII-02
ENr313_DpnII_Anchor2_Near
CCCCTGCAATACTCTCCAATTCATACACAT
hDpnII-03
ENr313_DpnII_Anchor1
ATGATACCTGTGTAGACCCAAGAAACCAGG
hDpnII-04
ENr313_DpnII_Anchor1_Near
GCTGTGATAAGCTTGGGAGAAGAGGTAATT
Primer PairB
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Day 4
7.3.2)
Digest the PCR products with MboI and ClaI as follows:
PCR product
10X Cutsmart
buffer*
MboI
ClaI
MilliQ
*NB!
Control (µl)
16.0
2.0
MboI (µl)
16.0
2.0
ClaI (µl)
16.0
2.0
MboI/ ClaI (µl)
16.0
2.0
----2
1
--1
--1
1
1
1
---
Using MboI instead of DpnII at this step enables using cutsmart buffer in a double digest with
MboI and ClaI
For HindIII Hi-C we use used NheI endonuclease to assess ligation efficiency (see Belton et al.,
Methods 2012).
7.3.3)
Incubate samples at 37°C for at least 30 minutes: Run digestion reactions on a long 2.5 %
agarose gel. Only successful biotin fill-in of DpnI sites (GATC) followed by blunt-end ligation
will have created a ClaI restriction site (ATCGAT).
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Day 6
8. REMOVAL OF BIOTIN FROM UN-LIGATED ENDS
8.1)
Set up for 40 µg total DNA as follows:
INGREDIENT
Hi-C DNA sample
10X NEBuffer 2.1
10 mM dATP
10 mM dGTP
3,000 U/ml T4 DNA polymerase
MILLIQ
8.2)
8.3)
1X
5 µg
5 µl
0.125 µl
0.125 µl
5 µl
TO 50 µL
NX
8.1.1) Split into 50-100 µl reactions to accommodate incubation in a PCR machine
8.1.2) Incubate at 20°C for 4 hours
Inactivate the enzyme for 20 mins at 75°C
Cool down/ keep at 4°C
N.B :In the final mix we have DNA, the reagents used in this step, and free nucleotides including the biotin-14dATP. In order to clean DNA for sonication we wash the DNA using amicon column
8.4)
Pool the reactions and perform an Amicon protocol:
8.4.1) Centrifuge at 14,000xg for ~ 5 mins or until volume has reduced to ~ 100 µl
8.4.2) Wash twice with 400 µl of milliQ
8.4.3) Invert the Amicon column onto a fresh tube and centrifuge for 2 minutes at 14,00xg to
collect the DNA
8.4.4)
NB!
Beware that Amicon does not remove proteins and that concentration by volume reduction often results
in DNA coming out of solution
8.5)
Add milliQ to a total of 130 µl and transfer to a Covaris microTUBE for sonication
9. DNA SONICATION
9.1)
Shear the DNA to a size of 200-300 bp using a sonicator. For a Covaris instruments use the following
parameters at 20°C:
Covaris model
S2
M220
E220 (evo)
Peak Incident Power (Watt)
---
50W
175W
Duty Cycle/Duty factor
10%
20%
10%
Intensity
5
----
---
Cycles per Burst
200
200
200
Treatment time (seconds)
4x 60
240
240
Set Mode
Frequency sweeping
Pre-use degassing
60 minutes
---
60 minutes
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Day 6
10. SIZE FRACTIONATION USING AMPURE XP
The liquid phase of AMpure mixture precipitates DNA onto the AMpure XP magnetic beads. The size of
the DNA molecules precipitated depends on the ratio of the volume of AMpure XP liquid (PEG8000) to
the volume of the sample when mixed together. Increasing the proportion of AMpure XP liquid decreases
the cutoff of the size of the DNA molecules precipitated on to the beads.
10.1)
10.2)
Bring volume of Hi-C sample up 500ul with 1x TLE
Allow AMpure XP mixture to come to RT and mix well prior to use. Add 400 µl of AMpure XP
mixture. Label the tube as 0.8X (the ratio of AMpure XP solution volume to sample volume is
400/500 = 0.8).
NB!
Under these conditions the beads capture and remove DNA fragments >350 bp from the sample.
10.3)
10.4)
10.5)
Vortex and spin down tubes briefly.
Incubate tubes for 10 min at RT.
Place tubes on the Magnetic Particle Seperator (MPS) for 5 min at RT.
10.5.1) During above incubation, prepare a fresh tube with 500 µl AMpure XP mixture; label them
as 1.1X.
10.5.2) Incubate tubes on the MPS for 5 min
10.5.3) Remove supernatant
10.5.4) Resuspend beads in 150 µl AMpure mixture
NB!
This step increases the number of beads present in a smaller volume of AMpure XP mixture. This
prevents saturation of the beads with DNA, ensuring an efficient precipitation of the DNA.
10.6)
Collect the 0.8X supernatants from MPS
10.6.1) Add the supernatants to the 1.1X tubes.
NB!
The ratio of AMpure XP solution volume to the original sample volume is now: (150 + 400)/500 = 1.1).
Under these conditions beads bind DNA fragments >100 bp.
10.7)
10.8)
10.9)
10.10)
10.6.2) Vortex and spin down tubes briefly.
10.6.3) Incubate 1.1X tubes for 10 min at RT
10.6.4) Place 1.1X tubes on the MPS for 5 min at RT
Discard supernatant from the 1.1X tubes.
Wash the beads in 0.8X and 1.1X tubes twice with 1 ml 70% ethanol, reclaiming beads against the
MPS for 5 min each time.
Air dry beads on the MPS until alcohol has evaporated completely
Resuspend the 0.8X and 1.1X beads in 50 µl of 1x TLE buffer in each tube to elute the DNA
10.10.1) Incubate 10 min at RT
10.10.2) Separate AMpure beads from eluate for both 0.8X and 1.1X tubes on the MPS for 5 min
10.10.3) Keep the eluate
(Optional) Check the quality and estimate the quantity of DNA on a 2% agarose gel. Run one lane of
the 0.8X sample and a dilution series of the 1.1X sample (for instance, 7, 3 and 1 µl of eluate) along
with a low molecular weight DNA marker. Quantify the amount of DNA in the 1.1X sample.
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Day 6
11. END REPAIR
11.1)
Add to the 50µl 1.1X Hi-C sample, in the following order for end-repair:
Ingredient
Size selected DNA eluate
10X ligation buffer [NEB]
25 mM dNTP mix
T4 DNA polymerase (3U/ µl)
T4 polynucleotide kinase (10U/ µl)
Klenow DNA polymerase I (5U/ µl)
Water
Total
1x
50 µl
7 µl
0.7 µl
2.5 µl
2.5 µl
0.5 µl
6.8 µl
70 µl
Nx
Nx 70µl
Incubate the reaction in a PCR machine:
11.1.1) 30 min at 20°C
11.1.2) 20 mins at 75°C to deactivate the enzymes
11.1.3) keep at 4°C(∞) until proceding to biotin pulldown
12. BIOTIN PULLDOWN WITH STREPTAVIDIN COATED BEADS
NB! All subsequent steps are performed in 1.7ml DNA LoBind tubes (Eppendorf) with DNA LoBind tips. Samples
are transferred to new tubes to prevent the loss of the beads on the tube wall.
12.1)
12.2)
12.3)
12.4)
12.5)
12.6)
12.7)
12.8)
12.9)
12.10)
12.11)
12.12)
Vortex the MyOne™ Streptavidin C1 beads and transfer 2 µl of bead solution per 1 µg of Hi-C DNA to
a 1.7ml LoBind tube with a minimum of 10 µl.
Wash the beads with 400 µl of Tween wash buffer (TWB) by pipetting up and down and incubating
for 3 min at RT on a rocking platform.
Reclaim beads against the MPS for 1 min, discard the supernatant.
Resuspend beads in 400 µl of TWB and transfer to a new LoBind tube.
Reclaim beads against the MPS for 1 min, discard the supernatant.
Resuspend beads in 400 µl of 2X Binding Buffer (BB)
12.6.1) Add 330 µl TLE
12.6.2) Add 70 of End-repair reaction containing DNA
12.6.3) Incubate the sample for 15 min at RT with rotation.
Reclaim the beads against the MPS for 1 min; discard the supernatant.
Resuspend the beads in 400 µl of 1X BB and transfer them to a new tube
Reclaim the beads against the MPS for 1 min, discard the supernatant.
Wash beads with 100 µl of 1x TLE (pH 8.0) and transfer to a new tube
Reclaim the beads against the MPS for 1 minute; discard the supernatant
Finally resuspend the beads in 41 µl of 1x TLE (pH 8.0)
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13. A-TAILING
13.1)
Mix components as follows:
INGREDIENT
Ampure purified DNA
10X NEBuffer 2.1
10 mM dATP
Klenow fragment (3’ → 5’ exo-) (5U/ µl)
TOTAL
13.2)
13.3)
13.4)
13.5)
13.6)
13.7)
1 LIBRARY
41 µl
5 µl
1 µl
3 µl
50 µL
N LIBRARIES
NX 50 µL
Incubate reactions in a PCR machine at 37°C for 30 min to add adenine on the 3’ end and then at 65°C
for 20 min to inactivate Klenow (exo-).
Place tubes on ice immediately.
Reclaim beads against the MPS for 1 min, discard the supernatant.
Resuspend the beads in 400 µl 1X ligation buffer (made of 5X T4 DNA ligase buffer [Invitrogen])
Reclaim the beads against the MPS for 1 minute; discard the supernatant
Finally resuspend the beads in 40 µl 1X ligation buffer1 (made of 5X T4 DNA ligase buffer
[Invitrogen])
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Day 6
14. ILLUMINA ADAPTER LIGATION AND PAIRED END PCR
14.1)
Set up one adapter ligation reaction as follows:
INGREDIENT
Illumina paired end adapter* (15 µM)
Hi-C DNA bound to streptavidin beads in 1X ligation buffer
5X T4 ligation Buffer [Invitrogen]
T4 DNA ligase [Invitrogen]
TOTAL
NB!
14.2)
14.3)
14.4)
14.5)
14.6)
14.7)
14.8)
14.9)
14.10)
14.11)
1 LIBRARY
5 µl
40 µl
2 µl
3 µl
50 µL
N LIBRARIES
NX 50 µL
*PAIRED-END ADAPTERS are constructed by annealing in a PCR machine
Incubate at 22˚C (RT) for 2 hours.
Reclaim the beads against the MPS for 1 min. Discard the supernatant.
Wash the beads twice with 400 µl of TWB. Add buffer to the beads, mix carefully by pipetting and
incubate at RT for 5 min with rotation
Reclaim the beads against the MPS for 1 min. Discard the supernatant.
Resuspend the beads in 200 µl of 1X BB and transfer to a new tube
Reclaim the beads against the MPS for 1 minute. Discard the supernatant.
Wash the beads twice with 200 µl of 1X NEBuffer 2.1. Resuspend beads then transfer to a new tube
Reclaim the beads against the MPS for 1 min. Discard the supernatant.
After the last wash, resuspend the beads in 20 µl of 1X NEBuffer 2.1 and transfer to a new tube.
Set up PCR to titrate number of cycles as follows:
INGREDIENT
Bead-bound Hi-C DNA
G29_PE primer 1.0 (10 µM)
G30_PE primer 2.0 (10 µM)
25mM dNTP
10X PfuUltra buffer
milliQ
PfuUltra DNA polymerase
TOTAL
1 REACTION
1.5 µl
0.5 µl
0.5 µl
0.5 µl
3 µl
23.5 µl
0.5 µl
30 µL
N REACTIONS
N*50 µL
For this step, unmodified PE primers ordered from any primer vendor may be used.
Run the PCR program with pauses after different number of cycles:
STEP
TEMP
TIME
1
95°C
120 seconds
2
95°C
20 seconds
3
65°C
20 seconds
4
72°C
15 seconds
5
Go to 2 N times
total 6, 8, 10, 12 and 14 cycles
6
72°C
3 minutes
Take 5 µl aliquots after 6, 8, 10, 12 and 14 cycles
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Day 6
G29_PE Primer 1.0 (HPLC) AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT
G30_PE Primer 2.0 (HPLC) CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT
H23_TruSeq-PE1.0 (HPLC) CAAGCAGAAGACGGCATACGAGA*T
H24_TruSeq-PE2.0 (HPLC) AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACG*A
* Indicates phosphorothioate bond
14.12)
14.13)
Quantify the amount of DNA after each PCR cycle by running the collected aliquots on a 2% agarose
gel with a low molecular weight DNA ladder.
Build a calibration curve showing the DNA quantity vs. number of cycles.
Choose an optimal number of cycles and number of PCR reactions for the final amplification of the
library for deep sequencing. The cycle number should be chosen so that the PCR amplification is in the
linear range and the expected size distribution is preserved (over cycling will shift the size distribution
of the library towards higher molecular weight products). The number of PCR reactions should be
calculated to produce the amount of DNA desired for sequencing (usually 50-100 ng). It is always better
to reduce the number of PCR cycles while increasing the number of PCR reactions. The optimal amount
is usually 1-2 cycles below the lowest amount visible on gel.
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Day 7
15. PRODUCTION PCR
15.1)
15.2)
15.3)
15.4)
15.5)
15.6)
15.7)
15.8)
Perform PCR reactions on the remainder of bead-bound template DNA in 1X NEBuffer 2.1 (~18 µl =
12 reactions) with chosen conditions and # cycles
Pool all PCR reactions together
Separate the beads from the supernatant and reserve 1% of it for a gel check.
To remove primer dimers, purify the amplified Hi-C library from the supernatant using AMpure XP
beads as follows. Allow AMpure XP mixture to come to RT and mix well prior the use. Add 1.5x
volumes of AMpure XP mixture to the supernatant.
15.4.1) Vortex and spin down briefly.
15.4.2) Incubate for 10 min at RT.
15.4.3) Place on the MPS for 5 min at RT.
15.4.4) Discard supernatant.
15.4.5) Wash the beads twice with 1 ml of freshly made 70% ethanol
15.4.6) Air-dry the beads completely
15.4.7) Resuspend the beads in 30 µl of TLE buffer
Incubate the beads for 10 min at RT, tapping the tube every 1-2 min.
Collect the beads with the MPS for 5 min
Transfer the supernatant, containing the final Hi-C library, to a new tube.
Quantify the amount of DNA in the Hi-C library on the 2% agarose gel as above or by
spectrophotometry.
16. QUALITY CONTROL OF HI-C LIBRARY BY RESTRICTION DIGEST
16.1)
Digest a small aliquot of the final Hi-C library with ClaI to estimate the portion of molecules with
valid biotinylated junctions.
INGREDIENT
10x NEB Cutsmart buffer
milliQ
ClaI
Hi-C product
TOTAL
1X
1.5 µl
to 15 µl
1 µl
0.75 -10 ng
15 µL
NX
NX 15 µL
NB!
For HindIII Hi-C use NheI in NEBuffer 2.1
16.2)
16.3)
Allow to digest at 37°C for 30 minutes.
Run the entire volume of the reaction on a 2% agarose gel.
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Additional info
REAGENTS
BUFFERS
5X ligation buffer [Invitrogen]
250 mM Tris-HCl (pH 7.6)
50 mM MgCl2
5 mM ATP
5 mM DTT
25% (w/v) polyethylene glycol-8000MilliQ dH2O
to 50 ml
~10 ml
--- Store 50ml and 1ml aliquots at -80°C or at 4°C and add DTT/ATP fresh!
1X Lysis buffer
10 mM Tris-HCl (pH=8.0)
10 mM NaCl
0.2% Igepal CA-630 (NP40)
MilliQ dH2O
--- Store at 4°C
Stock
1 M (pH=8.0)
5M
10%
to 50 ml
50 ml
500 µl
100 µl
1000 µl
~48.4 ml
Tween Wash Buffer (TWB)
5 mM Tris-HCl (pH=8.0)
0.5 mM EDTA
1 M NaCl
0.05% Tween
MilliQ dH2O
Stock
1M (pH=8.0)
500 mM
5M
100%
to 100 ml
100 ml
500 µl
100 µl
20 ml
50 µl
~79.4 ml
2X Binding Buffer (BB)
10 mM Tris-HCl (pH=8.0)
1 mM EDTA
2 M NaCl
MilliQ dH2O
Stock
1 M (pH=8.0)
500 mM
5M
to 100 ml
100 ml
1 ml
200 µl
40 ml
~58.8 ml
5X Annealing Buffer (AB)
50 mM Tris-HCl (pH=7.5)
50 mM NaCl
0.5 mM EDTA
MilliQ dH2O
Stock
1 M (pH=8.0)
5M
0.5 M
to 25 ml
25 ml
1.25 ml
250 µl
25 µl
~23.5 ml
TLE (pH 8.0)
10 mM Tris-HCl
0.1 mM EDTA
MilliQ dH2O
Stock
1M
500 mM
100 ml
1000 µl
20 µl
~99 ml
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Additional info
OTHER REAGENTS AND EQUIPMENT
Reagent
Company
Catalog number
Halt Protease Inhibitor Cocktail (100X)
Adenosine 5'-triphosphate magnesium salt from
bacterial source (ATP) - 1G
Thermo
78429
Sigma
A9187-1G
Biotin-14-dATP
Invitrogen
19524-016
Biotin-14-dCTP
Invitrogen
19518-018
Proteinase K (Fungal)
Invitrogen
25530-031
DNA polymerase I, large (Klenow) fragment
NEB
M0210S
T4 DNA ligase 1U/ µl
Invitrogen
15224090
T4 DNA polymerase
NEB
M0203L
5X ligation buffer
Invitrogen
46300-018
T4 polynucleotide kinase
NEB
M0201
Invitrogen
P/N y90001
Klenow fragment (3’ → 5’ exo )
NEB
M0212L
Dynabeads® MyOne™ Streptavidin C1
Invitrogen
650.01
PfuUltra II Fusion DNA polymerase
Stratagene
600670
Agencourt AMPure® XP
Beckman Coulter
A63881
DNA LoBind tubes, 1.5 ml
Eppendorf
022431021
Dow Corning® high-vacuum silicone grease colorless
Sigma
Z273554-1EA
Dounce homogenizer, pestle A
Kimble Chase
885301-0002
Amicon® Ultra – 0.5ml 30K
Millipore
UFC5030BK
Low molecular weight DNA marker
NEB
N3233S
1 kb DNA marker
NEB
N3232S
Dynal MPC™-S Magnetic Particle Concentrator
Invitrogen
120.20D
Illumina PE adapter
IDT
Sequence order
Illumina PE Primer 1.0, PE Primer 2.0
IDT
Sequence order
NEBuffer 2.1
NEB
B7202S
37% formaldehyde(FA)
Sigma Aldrich
252549
DSG
Thermo Fisher
20593
EGS
Thermo Fisher
21565
5X T4 DNA ligase buffer
-
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Additional info
PROTOCOL FOR ANNEALING OF FORKED PE ADAPTER OLIGOS

Prepare oligos to 100 µM stock solution (we order 250 nmole HPLC purified; IDT)
INGREDIENT
G28_PE-ADAPTER_top (SBS3, oligoA) 100 µM
G27_PE-ADAPTER_bottom (SBS8, OligoB) 100 µM
5x Annealing Buffer (AB)
TOTAL
1X
20 µl
20 µl
10 µl
50 µL
NX
NX 50 µL
The annealing was performed in a PCR machine as suggested by the Illumina patent:




ramp at 0.5 ˚C/sec to 97.5˚C
hold at 97.5˚C for 150 seconds (2.5 minutes)
step at 97.5˚C for 2 seconds with a drop of 0.1˚C/seconds for 775 cycles
Keep at 4˚C afterwards
G28_PE-ADAPTER_top (HPLC)
G27_PE-ADAPTER_bottom (HPLC)
ACACTCTTTCCCTACACGACGCTCTTCCGATCT
/5PHOS/GATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
H10_TruSeq_Adapter-Index01
/5PHOS/GATCGGAAGAGCACACGTCTGAACTCCAGTCACATCACGAT
CTCGTATGCCGTCTTCTGCTTG
/5PHOS/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCGATGTAT
CTCGTATGCCGTCTTCTGCTTG
/5PHOS/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTTAGGCAT
CTCGTATGCCGTCTTCTGCTTG
/5PHOS/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTGACCAAT
CTCGTATGCCGTCTTCTGCTTG
/5PHOS/GATCGGAAGAGCACACGTCTGAACTCCAGTCACACAGTGAT
CTCGTATGCCGTCTTCTGCTTG
/5PHOS/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGCCAATAT
CTCGTATGCCGTCTTCTGCTTG
/5PHOS/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCAGATCAT
CTCGTATGCCGTCTTCTGCTTG
/5PHOS/GATCGGAAGAGCACACGTCTGAACTCCAGTCACACTTGAAT
CTCGTATGCCGTCTTCTGCTTG
/5PHOS/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGATCAGAT
CTCGTATGCCGTCTTCTGCTTG
/5PHOS/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTAGCTTAT
CTCGTATGCCGTCTTCTGCTTG
/5PHOS/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGGCTACAT
CTCGTATGCCGTCTTCTGCTTG
/5PHOS/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCTTGTAAT
CTCGTATGCCGTCTTCTGCTTG
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTC
TTCCGATC*T
H11_TruSeq_Adapter-Index02
H12_TruSeq_Adapter-Index03
H13_TruSeq_Adapter-Index04
H14_TruSeq_Adapter-Index05
H15_TruSeq_Adapter-Index06
H16_TruSeq_Adapter-Index07
H17_TruSeq_Adapter-Index08
H18_TruSeq_Adapter-Index09
H19_TruSeq_Adapter-Index10
H20_TruSeq_Adapter-Index11
H21_TruSeq_Adapter-Index12
H22_TruSeq _Adapter-Universal
* Indicates phosphorothioate bond


Dilute to 15 µM (40µM / 2.67) by adding 83 µl of 1x annealing buffer (133 µl total for 2.67x diluted)
Store at -20˚C
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