- Materials
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- 10 million white blood cells isolated from whole blood
- Consumables
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- Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
- NlaIII restriction enzyme with CutSmart Buffer (NEB, R0125L)
- ECOSURF EH-9 (Dow, 64366-70-7)
- Glycine (Sigma, 56-40-6)
- Formaldehyde at 36.5% v/v (Sigma, 33220)
- IGEPAL CA-630 (Sigma, I8896)
- Protease Inhibitor Cocktail (Sigma, P8340)
- Chilled 10X phosphate-buffered saline (PBS) (Thermo Fisher, 70011044)
- Sodium dodecyl sulfate (SDS) at 10% v/v (Sigma, 71736)
- 5 M NaCl (Sigma, 71386)
- 1 M Tris-HCl pH 8.0 (Thermo Scientific, 15893661)
- Ziplock bags
- 0.2 µm filter
- 50 ml centrifuge tubes
- 2 ml Eppendorf DNA LoBind tubes
- 1.5 ml Eppendorf DNA LoBind tubes
- Equipment
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- Class I hood with active charcoal filter
- Temperature-controlled centrifuge
- Thermal cycler or heat block
- Thermomixer
- Vortex mixer
- Hula mixer (gentle rotator mixer)
- Ice bucket with ice
- P1000 pipette and tips
- P100 pipette and tips
- P200 pipette and tips
- P20 pipette and tips
- Wide-bore pipette tips
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Day 1: Pore-C experiment overview
During day 1, the PBMCs are prepared for stabilising of the three-dimensional interactions of DNA in the nucleus by chemically cross-linking DNA and protein. The nuclei are then permeabilised to expose the crosslinked cytoskeleton cage and nuclear structures before the chromatin is denatured. The DNA is now accessible to the chosen restriction enzyme† which passively diffuses through the crosslinked cytoskeleton cage and nuclear structures to digest the genome at compatible recognition sites. The sample is incubated overnight which creates clusters of DNA fragments held in proximity by crosslinks between DNA and the cytoskeleton, preserving the original interactions which were crosslinked.
†This protocol has been written using NlaIII and the heat denaturation method as our investigations have found this 4-cutter is particularly suitable for Pore-C across many different species, yielding Pore-C extracts with high contact densities. For more information, please see the "Protocol considerations" section of our Restriction enzyme Pore-C info sheet.
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Thaw the NlaIII restriction enzyme and CutSmart Buffer in accordance with the manufacturer's instructions and place on ice.
- Thaw both reagents on ice.
- Flick and/or invert the reagent tubes to ensure they are well mixed.
Note: Do not vortex the NlaIII restriction enzyme. - Spin down tubes before opening for the first time each day.
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Prepare 1 ml of 1% SDS in nuclease-free water, as follows:
Reagent Volume 10% SDS 100 µl Nuclease-free water 900 µl Total 1,000 µl -
Prepare 10 ml of 10% (v/v) ECOSURF™ EH-9 in nuclease-free water, as follows:
- Weigh out 1 g of ECOSURF™ EH-9.
- Transfer to a fresh 15 ml Falcon tube.
- Add 9 ml of nuclease-free water.
- Gently pipette mix with a wide-bore pipette tip until the solution is homogenous.
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Prepare 1 ml of 2.5 M glycine filtered through a 0.2 µm filter and store at room temperature.
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Prepare 200 ml filtered 1X PBS and chill at 4°C.
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Pre-cool a centrifuge to 4°C.
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Prepare the formaldehyde solution as follows:
Transfer 10 ml of 1X PBS into a 50 ml Falcon tube.
Note: Using a 15 ml Falcon tube is not recommended.Inside a class I hood, with double gloves, add 291 μl of 36.5% formaldehyde to the 10 ml 1X PBS aliquot to a final concentration of 1% formaldehyde in ~10.3 ml.
Mix by gentle inversion, and open the tube to allow gases to escape, then close the tube.
Check that no formaldehyde residue has remained on the gloves, Falcon tube, or pipette.
Remove the outer gloves and discard them in a biohazard bag in the hood.
Remove the 1% formaldehyde 1X PBS solution from the hood.
Store the tube with formaldehyde inside a zip lock bag at 4°C prior to use.
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Prepare the white blood cells as follows:
Take approximately 10 million white blood cells and briefly homogenise the suspension by gently pipetting with a wide-bore pipette tip.
Transfer the cell suspension to a 50 ml centrifuge tube.
Rinse the original tube with a further 1 ml of chilled 1X PBS into the 50 ml centrifuge tube.
Bring the volume of the resuspended white blood cells to 10 ml in chilled 1X PBS.
Proceed with the Pore-C experiment using approximately 10 million white blood cells as input.
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Centrifuge the sample at 300 x g at 4°C for 5 minutes.
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Aspirate and discard the supernatant, then add 10 ml of chilled 1X PBS to the pellet. Resuspend the pellet by gently pipetting up and down using a wide-bore pipette tip.
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Centrifuge the sample at 300 x g at 4°C for 5 minutes.
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Check the 2.5 M glycine solution has not precipitated before crosslinking the sample. Dissolve precipitate with heat and vortexing if required.
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Inside a class I hood, with double gloves, aspirate and discard the supernatant.
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Add 1 ml of the previously prepared 1% formaldehyde solution 1X PBS to the pellet. Resuspend the pellet by gently pipetting up and down using a wide-bore pipette tip.
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Once resuspended, add a further 9 ml of the 1% formaldehyde solution in 1X PBS. Mix gently by pipetting up and down, using a wide-bore pipette tip.
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Incubate at room temperature for exactly 10 minutes to crosslink the sample. The incubated solution should be mixed by gentle agitation every few minutes.
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Inside the hood with double gloves, quench the formaldehyde by adding 527 μl of 2.5 M glycine to the sample suspension for a final concentration of 1% w/v glycine (125 mM) in ~10.5 ml. Mix gently by pipetting up and down, using a wide-bore pipette tip.
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Incubate at room temperature for 5 minutes, then chill on ice for a further 10 minutes with regular, gentle agitation.
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Centrifuge the crosslinked sample suspension at 300 x g at 4°C for 5 minutes.
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Continuing in the class I hood, aspirate and discard the supernatant. Add 10 ml of chilled 1X PBS to the tube.
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Centrifuge the sample at 500 x g at 4°C for 5 minutes.
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Continuing in the class I hood, aspirate and discard the supernatant, and add 1 ml of chilled 1X PBS to the pellet. Mix gently by pipetting up and down using a wide-bore pipette tip.
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Split the resuspended sample into two separate 500 μl aliquots in fresh 2 ml Eppendorf tubes.
Note: 2 ml Eppendorf tubes are required for a compact sample pellet. Do not use 1.5 ml tubes.
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Wash the previous sample tube with a further 1 ml of 1X PBS, and split this between the two aliquots in 2 ml Eppendorf DNA LoBind tubes.
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Centrifuge the samples at 500 x g at 4°C for 5 minutes. Aspirate and discard the supernatant.
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Optional actionWe advise continuing with a freshly crosslinked sample pellet. However, if you intend to store samples for later use, you can snap-freeze the aliquots in liquid nitrogen. Store frozen sample pellets at –80°C and use within one year.
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Pre-cool a microfuge to 4°C and set a thermomixer to 65°C.
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Prepare 600 μl of 1.5X CutSmart Buffer in nuclease-free water as follows in a 1.5 ml Eppendorf DNA LoBind tube. Keep on ice.
Reagent Volume Nuclease-free water 510 µl 10X CutSmart Buffer 90 µl Total 600 µl -
To make the permeabilisation solution, add the components below to a 1.5 ml Eppendorf DNA LoBind tube in the following order. Keep the prepared permeabilisation solution on ice at 4°C until ready to use.
Reagent Final Volume Tris-HCl, pH 8.0, 1 M 10 mM 5 µl NaCl, 5 M 10 mM 1 µl IGEPAL CA-630, 10% 0.2% 10 µl Nuclease-free water - 484 µl Total - 500 µl -
Thaw the protease inhibitor cocktail on ice and spin down.
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Add 50 μl of protease inhibitor cocktail to 500 μl of permeabilisation solution at 4°C.
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Add 550 μl protease inhibitor cocktail-permeabilisation solution to the sample pellet. Resuspend the pellet by gently pipetting up and down, using a wide-bore pipette tip.
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Incubate on ice for 15 minutes and mix by regular, gentle inversion.
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Centrifuge the sample at 500 x g at 4°C for 10 minutes.
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Resuspend the pellet in 200 μl of the prepared chilled 1.5X CutSmart buffer by gently pipetting up and down, using a wide-bore pipette tip.
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Centrifuge the sample at 500 x g at 4°C for 5 minutes. Aspirate and discard the supernatant.
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Resuspend the pellet in 300 μl of the prepared chilled 1.5X CutSmart buffer by gently pipetting up and down, using a wide-bore pipette tip.
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To denature the chromatin, add 33.5 μl 1% SDS directly to the sample suspension to a final concentration of 0.1% SDS and a total volume of 333.5 μl. Mix gently by pipetting up and down using a wide-bore pipette tip.
The SDS may precipitate at this point; this will not impact the experiment so proceed to the next step.
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Incubate the sample suspension in a thermomixer at 300 RPM at 65°C for 10 minutes.
Note: This incubation can be performed without mixing.
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Remove the tube from the thermomixer and immediately put on ice.
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Set the thermomixer to 37°C.
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Add 37.5 μl of 10% (v/v) ECOSURF™ EH-9 directly to the cell suspension for a final concentration of 1% ECOSURF™ EH-9 (total volume of 371 μl). Mix gently by pipetting with a wide-bore pipette tip.
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Incubate the tube on ice for 10 minutes.
The SDS may precipitate at this point. This will not impact the experiment so proceed to the next step.
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Add the following reagents to the sample suspension and invert 3-4 times to mix.
Reagent Final Volume Permeabilised cells - 371 µl NEB NlaIII, 10 U/µl 1 U/µl 45 µl Nuclease-free water - 34 µl Total - 450 µl -
Incubate the tube in a thermomixer at 37°C for 18 hours with periodic <1000 rpm rotation for <30 seconds every 15 minutes. This will prevent condensation inside the lid.
Note: This incubation can be performed without mixing.