Feb 20, 2026
Spatial Hi-C
This protocol is a draft, published without a DOI.
- Zhenping Chen1
- 1Guangzhou National labratory
Protocol Citation: Zhenping Chen 2026. Spatial Hi-C. protocols.io https://protocols.io/view/spatial-hi-c-hefsb3bnf
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 06, 2025
Last Modified: February 20, 2026
Protocol Integer ID: 231634
Keywords: spatial architecture of chromatin, chromatin ligation, slide chromatin ligation, wide chromatin conformation, chromatin, regulatory roles within complex tissue microenvironment, spatial hi, capturing genome, resolved contact map, contact map, tissue section, spatial barcoding, complex tissue microenvironment, generation sequencing, intact tissue section
Abstract
This protocol describes spatial Hi-C, a method for capturing genome-wide chromatin conformation directly within intact tissue sections. Unlike conventional Hi-C, which relies on tissue dissociation, this approach preserves spatial architecture by performing in situ Hi-C reactions directly on tissue sections. The step-by-step workflow covers on-slide chromatin ligation, spatial barcoding, and library preparation for next-generation sequencing, providing a complete guide to generating spatially resolved contact maps. This method opens new avenues for investigating the spatial architecture of chromatin and its regulatory roles within complex tissue microenvironments.
Attachments
Materials
| A | B | C | |
| REAGENT OR RESOURSE | SOURCE | IDENTIFIER | |
| Chemicals, Peptides, and Recombinant Proteins | |||
| Biotin-14-dATP | Life Technologies | cat#19524016 | |
| MboI | New England Biolabs | cat#R0147L | |
| DNA Polymerase I, Large (Klenow) Fragment | New England Biolabs | cat#M0210L | |
| T4 DNA Ligase | New England Biolabs | cat#M0202L | |
| Dynabeads MyOne Streptavidin C1 | Life Technologies | cat#65002 | |
| NEBuffer 3.1 | New England Biolabs | cat#B6003S | |
| Recombinant RNase Inhibitor | Takara | cat#2313B | |
| SUPERase·InRNase Inhibitor | Invitrogen | cat#AM2694 | |
| Nuclease free water | Invitrogen | cat#AM9932 | |
| NovoNGS® Tn5 Transposase | Novoprotein, Shanghai, China | cat#M145 | |
| OCT | Sakura | cat#4583 | |
| Flash gel | Lonza | cat#57031 | |
| Digitonin | Beyotime Biotechnology | cat#ST1272(25mg) | |
| Proteinase K | Thermo fisher | cat# EO0491 | |
| Critical Commercial Assays | |||
| ChIP DNA Clean & Concentrator Kit | Zymo | cat#D5205 | |
| NEBNext High-Fidelity 2X PCR Master Mix | New England Biolabs | cat#M0541L | |
| KAPA HiFi HotStart ReadyMix | Roche | cat#KK2601 | |
| PDMS microfluid chips (20μm,10μm) | Suzhou HiComp Microtech technology Co., Ltd. | custom | |
| PDMS microfluid chips (50μm) | Suzhou cchip scientific instrument Co., Ltd. | custom | |
| Hematoxylin and Eosin Staining Kit | Beyotime Biotechnology | cat#C0105S | |
| Slide for 50μm assay | Citotest Scientific | cat#188105 | |
| Slide for 20μm & 10μm assay | Haimen district Schleder | custom |
The key reagents in this protocol
Troubleshooting
Fixation, digestion and proximity ligation
Take the section out of the -80°C freezer and leave them at room temperature for 20 min.
The tissue slide was washed by 1 mL PBS twice to remove the OCT，5 min each.
The tissue slide was then fixed with 1% formaldehyde (27.8 μL 37%formaldehyde in 1 mL PBS) for 10 min.
Aspirate the formaldehyde solution from the section into an EP tube. Add 81 μL of 2.5 M Glycine to the EP tube, mix well, then add the mixture back onto the section and incubate at room temperature for 5 min.
The tissue slide was washed twice with 1 mL PBS, cleaned in deionized water and dried by air.
A bright-field image was taken using a 10 × objective (Thermo Fisher Scientific, EVOS M7000 microscope) as the Fix image.
Assemble the slide, PDMS glasket and the clamp together.
Add 200 μL lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl₂, 0.01% Tween-20, 0.01% NP-40, 0.001% digitonin and 1% BSA) to the PDMS glasket well, incubate at room temperature for 15 min.
Aspirate and discard the lysis buffer, add 200 μL wash buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl₂, 1% BSA and 0.1% Tween-20) to the PDMS glasket well, incubate at room temperature for 5 min.
Aspirate and discard the lysis buffer, add 50 μL 0.5% SDS to the PDMS glasket well, incubate at 62°C for 10 min.
Aspirate the SDS solution from the section into an EP tube. Add 145 μL of Nuclease-free water and 25 μL of 10% Triton X-100 to the tube, mix well then add the mixture back onto the section and incubate at 37°C for 15 min.
Aspirate the mix in the glasket well to an EP tube. Add 25 μL of 10 × NEB buffer 2 and 200 U of MboI to the tube, mix well then add the mixture back onto the section and incubate at 37°C for 7 h.
The tissue section was incubated at 62°C for 20 min to inactivate the enzyme and then cooled to room temperature.
Aspirate and discard the mixture, wash the tissue section twice by 1 × NEB buffer 2, 2 min eah.
Aspirate and discard the wash buffer, add 45 μL of fill-in master mix (37.5 μL of 1 × NEB buffer 2, 5.625 μL 0.4 mM biotin-14-dATP, 0.675 μL of 10 mM dCTP/dGTP/dTTP mix and 1.2 μL of 5 U/μL DNA polymerase I, large) to the glasket well and incubated at 37°C for 1.5 h.
Aspirate the fill-in master mix to a EP tube. Add Ligation master mix (100.35 μL of NF water, 18 μL of 10 × NEB T4 DNA ligase buffer, 15 μL of 10% Triton X-100 and 0.9 μL of 20 mg/mL Recombinant Albumin and 1.5 μL of 400 U/μL T4 DNA ligase) to the tube, mix well then add the mixture back to the section and incubate at 16°C overnight.
Tn5 transpsome assemble
100 μM HiC-5-reads 1 and HiC-3-reads 2 oligos were respectively annealed with an equal amount of
100 μM Blocked ME oligo by heating at 95°C for 5 min and slowly cooling down to 20°C at a ramp rate of
−1°C/min.
The annealed oligos were mixed with an equal volume. Then the transposome was assembled following the manufacturer’s guidelines, the transposome can be stored at -20℃.
Tn5 transposition
Aspirate and discard the wash buffer, add 200 μL 0.1 N HCl and incubate at room temperature for 5 min. followed by washed twice with PBS again.
Aspirate and discard the buffer, wash the tissue section twice with 1 × PBS, 5 min each.
Aspirate and discard the wash buffer, add 50 μL transposition mix (25 μL 2 × Tagmentation buffer, 16.5 μL 1 × DPBS, 0.5 μL 10% Tween-20, 0.1 μL 5% digitonin and 8 μL transposome) to the PDMS glasket well and incubate at 37°C for 40 min.
Aspirate and discard the transposition mix, add 200 μL stop buffer(40 mM EDTA) to the PDMS glasket well incubate at room temperature for 5 min to stop transposition.
Aspirate and discard the stop buffer, wash the tissue section with 200 μL 1 × NEB buffer 3.1 for 5 min.
Aspirate and discard the wash buffer, disassemble the clamp and the PDMS glasket well from the slide. Then, the tissue slide was dipped in deionized water for quickly cleaning and then dried with air.
Barcodes Ligation
There were two rounds of barcode ligation in spatial Hi-C, each round uses different 50 or 96 barcodes.
For spatial Hi‑C barcode stock preparation, barcode A (10 μL, 100 μM) and Hi‑C linker 1 (10 μL, 100 μM) were combined with 20 μL of 2 × annealing buffer (20 mM Tris, pH 7.5–8.0, 100 mM NaCl, 2 mM EDTA) and mixed well. The same procedure was followed for barcode B with Hi‑C linker 2. The mixtures were then heated at 95 °C for 5 min and slowly cooled to 20 °C at a ramp rate of −1 °C/min. The annealed products were stored at −20 °C.
Barcode ligation mix preparation: 2 μL of ligation mix (72.4 μL of NF water, 27 μL of T4 DNA ligase buffer, 11 μL T4 DNA ligase, 5.4 μL of 5% Triton X-100), 2 μL of 1 × NEB buffer 3.1 and 1 μL of each annealed spatial Hi-C barcode A (A1–A50/96, 25 μM) were added to each well. The same procedure was followed for Barcode B. Store at 4℃. The solution was freshly prepared and used immediately on the same day.
For barcode A in situ ligation, the first PDMS chip was used to cover the region of interest, the bright-field image was taken using a 10 × objective (Thermo Fisher Scientific, EVOS M7000 microscope). The tissue slide and PDMS device were then clamped.
Add 3.5 μL barcode A ligation reaction solution (50/96 tubes) was loaded into each inlet. Apply the vacuum and pull the solution until all channels are full.
Incubate in the humidified chamber at 37℃ for 30 min.
Aspirate and discard the remaining barcode A mixture from inlets/outlets.
Add 3.5 μL blocking buffer (345.6 μL of NF water, 40 μL of 10× T4 ligation buffer, and 14.4 μL of 100 μM Hi-C blocking 1) to each inlet. Apply the vacuum and pull the solution until all channels are full.
Incubate in the humidified chamber at 37℃ for 20 min.
Aspirate and discard the remaining blocking buffer from inlets/outlets. Add 3.5 μL 1 × NEB buffer 3.1 to each inlet. Apply the vacuum and pull the solution to wash the channel for 5 min.
Aspirate and discard the remaining wash buffer from inlets/outlets. Apply the vacuum and pull the solution for 5 min until no visible liquid remains in any channel.
Disassemble the clamp and the chip A from the slide. Then the slide was quickly dipped in deionized water and dried with air.
For barcode B in situ ligation, the second PDMS chip with channels perpendicular to the first PDMS was attached to the dried slide carefully. After acquiring a bright-field image, the PDMS was pressed onto the tissue using the clamp. Barcode incubation, blocking and washing followed the barcode A ligation protocol.
Disassemble the clamp and the chip B from the slide. Then the slide was quickly dipped in deionized water and dried with air.
The final bright-field image of the tissue was taken using a 10 × objective (Thermo Fisher Scientific, EVOS M7000 microscope) .
Tissue digestion and reverse crosslink
For tissue digestion, the region of interest was covered with a square PDMS gasket well. Assemble the slide, PDMS glasket and the clamp together.
And 100 μL reverse cross-linking solution (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1%SDS, 200 mM NaCl and 0.4 mg/mL proteinase K) to the PDMS glasket well and incubate in a humidified chamber at 58°C for 2 h.
Collect the lysate into a 1.5 mL EP tube. Add the 20 μL extra lysis solution to wash out the reservoir and retrieve as much lysate as possible.
Incubate the EP tube at 65℃ overnight with rotation.
Biotin enrichment
Prepare 30 μL Dynabeads MyOne Streptavidin C1 beads per library: 30 μL of 10 mg/mL beads were prepared for biotin pull-down by washing twice with 400 μL of 1 × TWB (5 mM Tris-HCl, pH 7.5; 0.5 mM EDTA; 1 M NaCl and 0.05% Tween 20).
For library construction, the lysate was first purified using the ChIP DNA Clean Concentration Kit according to the manufacturer's instructions and eluted into 25 μL of NF water.
Mix the gap filling products with 50 μL of 2 × BB (10mM Tris-HCl, pH 7.5; 1 mM EDTA and 2 M NaCl) and centrifuged at 10,000 RPM for 1 min.
Add the supernatant to the beads, mix well and rotate at 30 RPM for 30 min at room temperature.
Put the tube on a magnet for 2 min to separate the beads.
Discard the supernatant, wash the beads twice by 400 μL 1 × TWB buffer at 55°C for 2 min with mixing at 500 RPM.
Wash the beads once by 400 μL 10mM Tris-HCl (8.0).
Put the tube on a magnet for 2 min to separate the beads. Resuspend the beads in 21 μL of 10 mM Tris-HCl (8.0).
Library construction
Add 21 μL beads to the PCR reaction system (25 μL of KAPA HiFi HotStart Ready Mix, 2 μL of Nextera i5 index primer (12.5 μM) and 2 μL of Nextera i7 index primer (12.5 μM)), mix well.
The samples were subjected to PCR amplification with the following procedure: 95°C for 3 min; 5 cycles of 98°C for 20 s, 60°C for 45 s, and 72°C for 1 min.
Place the tube on the magnet, take the supernatant into a new PCR tube, and PCR was performed again for another 3-5 cycles with final extension (72°C for 5min).
After amplification, purify the fragments ranged from 300 to 800 bp by 1.5% agarose gel.
DNA library was then quantified and sequenced using an Illumina sequencing platform.