Nov 18, 2020
Dip-C (Part 1: Chromosome Conformation Capture, for Fixed Nuclei)
- 1Stanford University
Protocol Citation: Longzhi Tan 2020. Dip-C (Part 1: Chromosome Conformation Capture, for Fixed Nuclei). protocols.io https://dx.doi.org/10.17504/protocols.io.bpt7mnrn
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 18, 2020
Last Modified: November 18, 2020
Protocol Integer ID: 44639
Protocol materials
Sodium dodecyl sulfate solution BioUltra for molecular biology 10% in H2OMerck MilliporeSigma (Sigma-Aldrich)Catalog #71736
Triton X-100, 10% solution Merck MilliporeSigma (Sigma-Aldrich)Catalog #93443
NEBuffer 2 (10X)New England BiolabsCatalog #B7002S
MboI (25,000 units/ml) - 2,500 unitsNew England BiolabsCatalog #R0147M
T4 DNA Ligase Reaction Buffer - 6.0 mlNew England BiolabsCatalog #B0202S
BSA, molecular biology grade, 20 mg/ml New England BiolabsCatalog # B9000S
T4 DNA Ligase (1 U/µL)Thermo FisherCatalog #15224025
Corning™ Falcon™ Test Tube with 35µm Cell Strainer Snap CapCorningCatalog #352235
Falcon 40 µm Cell StrainerCorningCatalog #352340
PBS, pH 7.4Thermo FisherCatalog #10010023
PBS, pH 7.4Thermo FisherCatalog #10010023
DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride)Thermo FisherCatalog #D1306
96 well LoBind PCR plates Semi-skirtedEppendorfCatalog #0030129504
Digestion
Digestion
40m
40m
Thaw a tube of fixed nuclei On ice .
Prepare 0.5% SDS (50 µL per sample; recipe below for 100 µL ):
- 5 µL Sodium dodecyl sulfate solution BioUltra for molecular biology 10% in H2OMerck MilliporeSigma (Sigma-Aldrich)Catalog #71736 (final: 0.5 Mass Percent )
- 95 µL water
- Vortex to mix
Resuspend nuclei in 50 µL 0.5% SDS.
Incubate at 62 °C for 00:10:00 .
10m
Add:
- 145 µL water
- 25 µL Triton X-100, 10% solution Merck MilliporeSigma (Sigma-Aldrich)Catalog #93443
Rotate at 37 °C for 00:15:00 .
15m
Add restriction enzyme and its buffer:
- 25 µL NEBuffer 2 (10X)New England BiolabsCatalog #B7002S
- 20 µL MboI (25,000 units/ml) - 2,500 unitsNew England BiolabsCatalog #R0147M
Rotate at 37 °C Overnight .
15m
Ligation
Ligation
40m
40m
Centrifuge at 1000 x g, 4°C, 00:05:00 .
Make Ligation Buffer (2 mL per sample; recipe below for 1 mL ):
- 100 µL T4 DNA Ligase Reaction Buffer - 6.0 mlNew England BiolabsCatalog #B0202S
- 5 µL BSA, molecular biology grade, 20 mg/ml New England BiolabsCatalog # B9000S
- 865 µL water
- Vortex to mix.
Remove supernatant leaving ~50 µL .
Resuspend in 1 mL Ligation Buffer.
Centrifuge at 1000 x g, 4°C, 00:05:00 .
Remove supernatant leaving ~50 µL .
Resuspend in 1 mL Ligation Buffer.
Add 10 µL T4 DNA Ligase (1 U/µL)Thermo FisherCatalog #15224025 .
Pipette to mix.
Incubate at 16 °C for 04:00:00 , occasionally inverting the tube.
4h
Optionally filter with Falcon 40 µm Cell StrainerCorningCatalog #352340 or Corning™ Falcon™ Test Tube with 35µm Cell Strainer Snap CapCorningCatalog #352235 to avoid clogging the flow cytometer.
Aliquot if needed.
Centrifuge at 1000 x g, 4°C, 00:05:00 .
Remove supernatant.
Store at -80 °C .
Flow Sorting
Flow Sorting
40m
40m
On the day of flow sorting, thaw a tube of ligated nuclei On ice .
Resuspend in 1 mL PBS, pH 7.4Thermo FisherCatalog #10010023 .
Make 300 uM DAPI:
- 100 µL PBS, pH 7.4Thermo FisherCatalog #10010023
- 2.1 µL 14.3 mM (5 mg/mL) DAPI (stock made by dissolving DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride)Thermo FisherCatalog #D1306 in 2 mL water and stored at 4 °C )
- Vortex to mix.
Add 1 µL 300 uM DAPI (final: 0.5 nanomolar (nM) ).
Pipette to mix.
Flow sort single nuclei into96 well LoBind PCR plates Semi-skirtedEppendorfCatalog #0030129504 either dry or containing lysis buffer (which requires lysis by incubation before storing; see Part 2 for details).
Proceed directly to Part 2, or store at -80 °C .