HiFi-C (High-Fidelity Chromosome Conformation Capture) is a high-fidelity chromatin conformation capture technology based on high-throughput sequencing. It captures the spatial interactions of chromatin within the nucleus, allowing for precise analysis of the three-dimensional structure of the genome (such as TADs and chromatin loops) and the interaction patterns of gene regulatory elements (promoter-enhancer interactions). This technology can be applied in areas such as chromosome mapping, studies of chromatin conformation abnormalities related to diseases, and comparative 3D genomics across species.
This technology has high requirements for the integrity of chromatin in samples and the absence of cross-contamination. The standardization of sample preparation and handling directly determines the success of the experiment, and strict adherence to this protocol is required.
1
Animal Cell Line Samples
1.1 Sample Requirements
(1) Sample Type: Adherent cells or suspension cells (must be logarithmic growth phase cells, avoid using senescent or apoptotic cells).
(2) Minimum Sample Volume: Frozen tube aliquots ≥1×10⁷ cells (viable cell ratio ≥90%)/tube, it is recommended to prepare one backup tube.
1.2 Sample Preparation Process
(1) Cell Collection Procedure:
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Adherent Cells: Discard the culture medium, wash the cells 1-2 times with pre-cooled (4℃) sterile PBS buffer, add an appropriate amount of trypsin (containing EDTA) to digest the cells, and after the cells detach, add serum-containing medium to stop digestion, centrifuge at 1000rpm, 4℃ for 5 minutes, discard the supernatant, remove as much residual medium as possible, and collect the cell pellet.
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Suspension Cells: Take the logarithmic growth phase cell suspension, centrifuge at 1000rpm, 4℃ for 5 minutes, discard the supernatant, and collect the cell pellet. Resuspend the cell pellet in pre-cooled (4℃) sterile PBS buffer, centrifuge again (1000rpm, 4℃, 5 minutes), discard the supernatant, and remove as much residual medium as possible (components such as serum proteins in the medium can interfere with subsequent experiments).
(2) Quality Control:
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Visual Inspection: The cell pellet should appear uniformly milky white, with no obvious flocculent material or foreign objects; if conditions permit, preliminary observation of cell density can be done under a microscope to ensure no significant aggregation (aggregated cells can lead to uneven lysis in subsequent steps).
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Subsequent Testing: Check the crosslinking effect to ensure the chromatin structure is suitable for subsequent experiments.
1.3 Storage and Transport
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Add an appropriate amount of cell freezing solution, slowly cool and freeze (4℃ for 10 minutes → -20℃ for 30 minutes → -80℃ for 16-18 hours (overnight); or use a controlled-rate freezer or programmable cooling box to cool at -1 to -3℃/min to -80℃), and store long-term at -80℃, recommended storage duration ≤1 month.
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During transport, use sufficient dry ice (for 48 hours of transport, dry ice amount ≥10 kg) to maintain a low-temperature environment, and isolate the dry ice from the samples with foam board.
2
Animal Tissue Samples
2.1 Sample Requirements
(1) Sample Type: Soft tissue samples (preferably liver, spleen, kidney, muscle, etc., avoid tough/keratinized tissues such as skin, tendons, etc.).
(2) Minimum Sample Volume: Tissue weight ≥2g, it is recommended to prepare one backup tube.
2.2 Sample Preparation Process
(1) Tissue Collection Procedure:
Within 15 minutes after dissection, use a scalpel to remove fat, connective tissue, blood vessels, and other impurities, and gently wash the surface of the tissue with pre-cooled (4℃) sterile PBS buffer to remove blood stains and contaminants, eliminating residual blood (blood components can interfere with chromatin extraction). Use sterile absorbent paper to dry the surface moisture of the tissue, and place it in a non-enzymatic freezing tube.
(2) Quality Control:
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Visual Inspection: The tissue should exhibit normal physiological coloration (e.g., liver should be reddish-brown, muscle should be bright red), with no signs of necrosis or decay.
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Subsequent Testing: Check the crosslinking effect to ensure the chromatin structure is suitable for subsequent experiments.
2.3 Storage and Transport
(1) Rapidly freeze the tissue in liquid nitrogen (ensure complete freezing within 30 minutes), then transfer to a -80℃ freezer for storage, with a recommended storage duration ≤1 month (exceeding this may lead to chromatin breakage).
(2) During transport, use sufficient dry ice (for 48 hours of transport, dry ice amount ≥10 kg) to maintain a low-temperature environment, and isolate the dry ice from the samples with foam board to prevent local frostbite.
3
Animal Blood Samples
3.1 Sample Requirements
(1) Sample Type: Whole blood.Do not use heparin sodium as an anticoagulant (heparin sodium can affect subsequent crosslinking, enzyme digestion, and other experimental steps), it is recommended to use Streck tubes (containing specific anticoagulants) or EDTA anticoagulant tubes.
(2) Minimum Sample Volume:
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Mammals (e.g., humans, rats, mice): 5-10mL/sample;
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Non-mammals (e.g., fish, birds, amphibians): 0.5-2mL/sample.
3.2 Sample Preparation Process
(1) Blood Collection Procedure:
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Use sterile blood collection techniques (e.g., orbital blood collection in mice, cardiac blood collection in rats), ensuring that blood collection instruments (needles, anticoagulant tubes) are sterilized and free from DNAse/RNAse contamination.
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Immediately after blood collection, inject the blood into the anticoagulant tube, gently invert 10 times to mix the blood with the anticoagulant thoroughly, preventing clotting (clotting can damage blood cell structure, leading to loss of chromatin integrity).
(2) Quality Control:
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Visual Inspection: The blood should appear uniformly red, with no clots or hemolysis (hemolysis can release intracellular impurities, interfering with subsequent experiments).
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Subsequent Testing: Check the crosslinking effect to ensure the chromatin structure is suitable for subsequent experiments.
3.3 Storage and Transport
(1) Storage Conditions: After blood collection, it must be kept upright at room temperature (15-25℃), freezing is strictly prohibited (freezing can cause red blood cells to rupture, leading to hemolysis).
(2) Transport Requirements:
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Pack in a foam box, wrap the anticoagulant tubes with shock-absorbing materials (e.g., bubble wrap), and place ice packs inside the box (ice packs must be isolated from the anticoagulant tubes to prevent local temperature extremes), maintaining a temperature of 4℃ inside the box.
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Transport duration must be ≤24h, to avoid prolonged exposure of blood outside the body leading to cell apoptosis and chromatin degradation.
4
Plant Tissue Samples
4.1 Sample Requirements
(1) Sample Type: Young leaves from seedlings after seed germination (preferably cotyledons or the first 1-2 true leaves, avoid old leaves, diseased leaves, or leathery/waxy leaves). If the leaves contain high polyphenols/polysaccharides (such as certain grasses), please inform the laboratory in advance to adjust the protocol.
(2) Minimum Sample Volume: Tissue weight ≥2g, it is recommended to prepare one backup tube.
4.2 Sample Preparation Process
(1) Tissue Collection Procedure:
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Immediately after leaf detachment, gently wash the surface with sterile deionized water to remove dust, microorganisms, and other impurities, avoiding rubbing the leaves (to prevent cell wall damage, which can release secondary metabolites that interfere with subsequent experiments).
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Use sterile absorbent paper to dry the surface moisture of the leaves, and remove any yellowed or damaged leaf tissue.
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Place the qualified leaves in non-enzymatic centrifuge tubes or clean sealed bags; if short-term (≤12h) storage is needed, place a small amount of moist sterile absorbent paper inside the sealed bag (leaves should not directly contact moisture to prevent mold growth).
(2) Quality Control:
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Visual Inspection: The leaves should be bright green, with no browning or mechanical damage.
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Subsequent Testing: Check the crosslinking effect to ensure the chromatin structure is suitable for subsequent experiments.
4.3 Storage and Transport
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Rapidly freeze the leaves in liquid nitrogen (ensure complete freezing within 30 minutes), then transfer to a -80℃ freezer for storage, with a recommended storage duration ≤1 month (exceeding this may lead to chromatin breakage and DNA degradation).
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During transport, use sufficient dry ice (for 48 hours of transport, dry ice amount ≥10 kg) to maintain a low-temperature environment, and isolate the dry ice from the samples with foam board to prevent local temperature extremes from damaging the samples.
5
Packaging and Transport Specifications
5.1 General Packaging Requirements
(1) Inner Packaging: Samples (centrifuge tubes/frozen tubes) must be placed in sealed self-sealing bags or 50mL centrifuge tubes, with cotton or tissue added for fixation (do not add dangerous goods such as liquid nitrogen inside the 50mL tube or other supports); if liquid samples (such as blood), additional parafilm sealing film should be used to seal the centrifuge tube cap to enhance sealing. Sample tube caps/tube walls and self-sealing bags must be marked with waterproof pens indicating sample information (sample number + sample name + tissue type + collection date).
(2) Outer Packaging: Use a foam box (specific size based on actual weight) as the transport container, filling the box with shock-absorbing materials (such as bubble wrap, foam blocks) to prevent damage to samples during transport due to vibration; place ice packs or dry ice according to sample storage temperature requirements (ice packs/dry ice must be isolated from the samples with foam board to prevent local temperature extremes).
5.2 Transport Precautions
(1) Courier Selection: Preferably choose SF Express (next-day delivery service) to ensure quick delivery of samples; for remote areas, confirm courier timing in advance to avoid delays.
(2) Accompanying Documents: A “Sample Information Sheet” must accompany the samples, indicating sample number, name, grouping, and other key information, and the sample name must match the information filled in the sample tube for laboratory verification and subsequent tracking.
Next Issue Preview
This series’ previous article introduced the effective data differences between HiFi-C and Pore-C when aligned to reference genomes. This article presents the HiFi-C sample submission guidelines, and the next article will introduce the HiFi-C chromosome mapping analysis process and software differences. Feel free to scan for inquiries.
Reminder: The HiFi-C Technology Exploration Series consists of 6 issues, and this is the 5th issue. Follow us to not miss any exciting content!
About Feisha Gene
Wuhan Feisha Gene Information Co., Ltd. (referred to as Feisha Gene) is a high-tech enterprise integrating innovative multi-omics research technology services, precision medical testing services, and product development. The company is committed to developing innovative high-throughput multi-omics and bioinformatics technologies, establishing a comprehensive technical platform that includes the latest second/third-generation high-throughput sequencing, genomics, 3D genomics, epigenomics, single-cell spatiotemporal omics, and bioinformatics. It has independently developed multi-omics experimental techniques and fully automated bioinformatics data analysis algorithms and processes for different application scenarios, providing research technology services and conducting research project collaborations for life science and medical researchers. In terms of genomic medicine products and services, the company focuses on the development and service of detection technology products related to precision medicine for major diseases. Since its establishment, the company has published collaborative research results in internationally renowned journals such as Cell, Nature, and Science, with over 600 papers published and a cumulative impact factor exceeding 5000. The company has applied for and obtained 62 national invention patents and over 200 software copyrights. Feisha Gene has been recognized as a national high-tech enterprise, a national gene testing application demonstration center, a national-level specialized and innovative “little giant” enterprise, a gazelle enterprise, a strategic team for innovation and entrepreneurship in Hubei Province, one of the first thousand enterprise support plans in Wuhan, and part of the “3551” Optics Valley Talent Program, among other supports and honors.
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