Hi-C

Hi-C / MetaPhase protocol   (Ivan Liachko, 2/2014)

Timeline:

Day0 – before HiC start, grow cells, crosslink, freeze cells

Day1 – cell lysis, digest overnight

Day2 – fill-in with biotin, ligation, reverse crosslinks overnight

Day3 – recover DNA, streptavidin immobilization, library prep (busy, takes all day)

Day4* - DNA recovery can use up much of Day3, sometimes Day4 is for library prep.

Collection and crosslink of cells:

Cultures are grown/diluted to OD600 of 1-2 (usually 500mL-1L in a 2L flask).

37% formaldehyde added to a final concentration of 1% (13.5mL/500mL culture).

Incubate at room temp for 20-30min with periodic swirling around.

Quench crosslink by adding glycine (5g/500mL culture). 

Swirl around to mix and let stand at room temp for 20-30min.

Spin down culture, rinse cells in same media and aliquot cells.

For yeast cultures, I end up with 2mL tubes with cell pellets of ~100uL dry volume. 

Decant the supernate and freeze pellets in -20 or -80

Cell Lysis

For yeast, a 100uL pellet is split into two 2mL tubes.  Each one is usually enough for a separate experiment (i.e. restriction digest).

Add ~500uL glass beads.

Add 1mL of 1xTBS with 1% Triton-X and Protease Inhibitors (no EDTA)

Keep on ice.  Vortex 5 times (each 5min vortex with 1-2min rest on ice in between)

Put each 2mL tube into a 15ml conical tube, so it hangs inside the bigger tube.

Poke a small hole at the bottom of 2mL tube with hot needle, also poke at the top.

The tube will drip into the 15mL conical.

Spin the assembly at 2-4KRPM for 2-5min to get all the liquid into the 15mL tube.

Transfer the flowthrough into a fresh 2mL tube (the pellet contains the good stuff).

Spin at 13KRPM for 10min to pellet all the chromatin and cell chunks.

Remove sup, add 1mL of same TBS/T to rinse pellet (gently mush with pipet tip).

Repeat the rinse with 1xTBS (no Triton this time).

Resuspend pellet in 500uL of 10mM Tris pH8 (like TE, but no EDTA)

At this point I like to quantitate the DNA even though it’s just a chunky mess.

QuBit = usually 1-10ng/uL

Digest chromatin (I usually do two separate enzyme digests, ex. HindIII and NcoI)

200 uL of chromatin suspension

120 uL of water

40 uL of NEBuffer X (depends on which enzyme used)

20 uL Restriction Enzyme

Incubate overnight at 37 degrees.

Fill-in DNA ends with biotin (at this step, using half of the digested chromatin is OK to save on biotinylated nucleotide costs, or just double the volume of everything)

250 uL of digested chromatin

Add 9 uL of each nucleotide dA, dT, dG diluted to 1mM prior to addition.

Add 20 uL of biotinylated dCTP at 0.4mM (Invitrogen, 19518-018)

Add 7.5 uL of Klenow (NEB)

Incubate at 37 degrees for 45-60minutes.

Inactivate enzymes by incubating at 70 degrees C for 10-15 minutes (not longer or crosslinks will start reversing).

Ligation

QuBit the chromatin to estimate DNA concentration. DNA concentrations in the ligation mix should be no higher than 0.5 ng/uL.

Set up 4mL ligations (if lots of DNA you can do 8mL, just double everything):

250 uL chromatin (or however much to get 0.5 ng/uL final concentration)

3340 uL water

400 uL T4 DNA Ligase buffer (NEB)

25 uL T4 DNA Ligase enzyme (NEB)

Incubate at room temp for 4-6 hours.

Reverse Crosslinks

Add proteinase K (50 uL of 10mg/mL for each 4mL total reaction)

Incubate at 70 degrees C overnight.

Purify DNA by phenol/chloroform, EtOH precipitation:

add 1 volume of phenol/chloroform, vortex for 30sec, spin for 5 minutes.

remove top layer into fresh tube, add 1 volume of chloroform, vortex, spin down.

Remove top layer, add 0.1volume of 3M NaoAc, 2 volumes of 100% EtOH, mix gently

Spin for 10 min at 13KRPM (split into 2mL epi tubes). Decant supernate.

Rinse pellets with 1 mL of 70% EtOH each.  Spin at 13KRPM for 10min again.

Decant and air dry pellets. 

Resuspend in combined volume of 600 uL water with RNAse A (6uL of 10mg/mL)

Incubate at 37 degrees for 30-60min to dissolve DNA and let the RNAse do its thing.

Remove biotinylated unligated ends

To 600uL of DNA

Add 66uL NEBuffer 2

6.6uL BSA (NEB)

6uL 10mM dATP (*not sure why, carryover from another protocol)

6uL 10mM dGTP (*not sure why, carryover from another protocol)

7.5uL T4 DNA Polymerase (NEB)

Incubate at room temp for 10min, then 12 degrees for 1 hour in the PCR machine.

Cleanup DNA via DNA Clean and Concentrate -5 Kit (Zymo)

Add 3mL of DNA Binding Buffer, mix

Split into 2 tubes, run through 2 columns total. Discard flowthrough.

Wash each column with 500uL of wash buffer.

Elute in 69uL of water from each column (138 total)

Used 2 uL for QuBit quantitation (usually 1-2ng/uL)

Put the rest into covaris tube (fits 130uL, Covaris, 520052)

Illumina Library prep (protocol from the downstream part of Illumina Mate-Pair Library prep, or TruSeq, the reagents/mixes all come from those kits)

Shear in Covaris machine to average fragment length of 300bp.

Settings for Covaris E210: 

Intensity – 4

Duty Cycle – 10%

Cycles/burst – 200

Time – 90sec

Sample volume – 130uL

Temp (C) – 7

Water level – 6

E210 Intensifier – pn500141

After shearing, purify the DNA with a single Zymo DNA clean and concentrator column, elute in 50uL water or elution buffer such as EB.

Streptavidin bead Immobilization (beads = Invitrogen Dynabeads, 650.01)

Resuspend magnetic streptavidin beads by shaking the bottle well

Transfer 20uL of beads into clean epi tube (LoBind tubes preferable)

Place the tube into magnetic gizmo for 1min, remove supernate.

Wash beads with 50uL of Bead Bind buffer, twice.

Resuspend the beads in 50uL Bead Bind Buffer (per each sample).

Add 50uL of sheared, cleaned DNA to 50uL of beads. 

Incubate at room temp for 15min with periodic finger flick mixing.

Quick spin and place on magnet for 1min, remove and discard the supernate.

Wash beads 4 times with 200uL of Bead wash buffer.

Wash beads 2 times with 200uL of Resuspension buffer.

End Repair

Add 60uL water to the beads and resuspend.

Add 40uL of End Repair mix, resuspend.

Incubate at 30 degrees for 30 minutes.

Wash beads 4 times with 200uL of Bead wash buffer.

Wash beads 2 times with 200uL of Resuspension buffer.

A-Tailing

Add 17.5uL of water to beads, resuspend.

Add 12.5uL of A-Tailing mix, resuspend.

Incubate at 37 degrees for 30 min

**no bead washing here, proceed directly to adaptor ligation steps

Adaptor Ligation

To the 30uL of DNA/bead mix from above:

Add 4uL of water.

Add 2.5uL of Ligation mix.

Add 1uL of DNA Adapter Index (such as AD005 for example)

Resuspend and incubate at 30 degrees for 10min.

Add 5uL of Ligation Stop buffer, or proceed directly to bead wash.

Wash beads 4 times with 200uL of Bead wash buffer.

Wash beads 2 times with 200uL of Resuspension buffer.

PCR Amplification

To the beads pelleted on the magnet:

Add 20uL water.

Add 25uL of PCR Master Mix.

Add 5uL PCR Primer Cocktail.

PCR program:

- 95 degrees for 10sec

- 12-15 cycles of the following:

        95deg for 10sec

        60deg for 30sec

        72deg for 30sec 

- 72 degrees for 3min

- 4 degrees forever

Place PCR/beads mix on the magnet, let stand for 1min, remove liquid to fresh tube.

(it’s worth saving the beads in case PCR fails)

PCR Purification (Standard AMPure protocol)

To 45uL of PCR reaction:

Add 30uL of resuspended AMPure XP beads.

Incubate at room temp for 2 minutes with periodic finger flick mixing.

Put tube into magnetic gizmo, let stand for 2min, remove/discard all liquid sup.

Add 200uL of fresh 70% EtOH, incubate for 30 sec at room temp, remove supernate.

Repeat EtOH wash.

Air Dry beads for 10-15min

Add 20uL of water to dry beads, resuspend, incubate at room temp for 5min.

Put back onto magnet, let stand for 2-5min.

Remove the supernate and KEEP IT (do not discard this sup, it’s the good stuff)

QuBit 2uL of the final eluate: >1ng/uL = victory.

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