Chromatin conformation capture is a technique whereby the three-dimensional structure of chromatin — the condensed complex of DNA and protein that comprise chromosomes — is stabilised through chemical crosslinking to preserve spatial organisation within the nucleus (Sati and Cavalli, 2017). These nuclear structures may then be interrogated through a variety of approaches to enable us to understand DNA interactions, proximity in sequence space, and the three-dimensional structures of the chromatin within the nucleus.

The Pore-C protocol has been developed based on research by Oxford Nanopore Technologies and published literature (Lieberman-Aiden et al., 2009; Comet et al., 2011; Belton et al., 2012; Gavrilov, Golov and Razin, 2013; Nagano et al., 2015; Belaghzal, Dekker and Gibcus, 2017; Ulahannan et al., 2019). The protocol employs an ‘in-nucleus’ chromatin conformation capture approach to stabilise chromatin interactions within the nucleus (Nagano et al., 2015). This method contains the full complement of chromosomes of each cell within its own crosslinked cage of cytoskeleton and other proteins. This approach is thought to preserve nuclear structures, enhancing recovery of interactions within each chromosome known as cis-chromosomal contacts, whilst reducing the frequency of interactions between chromosomes known as trans-chromosomal contacts.

This process is achieved through a series of chemical and biological manipulations over the course of several days (Figure 1):

Figure 1. The Pore-C protocol carries out these stages to manipulate and stabilise the chromatin interactions using in-nucleus chromatin conformation.

Day 1

Samples other than cell culture first require sample preparation as instructed in the appropriate Pore-C sample preparation protocol for the sample type. The three-dimensional interactions of DNA within the nucleus are stabilised by chemically crosslinking DNA and protein within the nucleus using formaldehyde. For plant samples, crosslinking occurs in the presence of a pressure vacuum to allow the formaldehyde to infiltrate the plant tissue. Following this process, proximal interactions of the DNA are preserved for the remainder of the protocol.

Note: This is the only point the protocol may be paused and the sample pellets snap-frozen in liquid nitrogen for use within a year.

Next, the nuclei are permeabilised to expose the crosslinked cytoskeleton cage and nuclear structures before the chromatin is then denatured using detergent and gentle heat. This process relaxes the chromatin but maintains proximal crosslinked interaction. Following chromatin denaturation, the DNA is accessible to restriction digestion. A suitably selected restriction enzyme is added to samples and passively diffuses through the crosslinked cytoskeleton cage and nuclear structures to digest the genome at compatible recognition sites.

Over the course of an overnight incubation, this process gradually creates clusters of DNA fragments, or monomers, held in proximity by crosslinks between DNA and the cytoskeleton. This preserves the original interactions that were stabilised at the time of crosslinking.

Day 2

After the overnight restriction digestion, the restriction enzymes must be inactivated. Depending on the choice of restriction enzyme, this may be achieved with either heat or chemical denaturation. This step prevents the enzymes re-digesting ligated products later in the protocol.

Following the overnight restriction digestion, the clusters of crosslinked DNA are ligated together in proximity. DNA ligase is added to the sample and passively diffuses through the crosslinked cytoskeleton cage to ligate the cohesive ends of proximal monomers into chimeric Pore-C polymers.

Once the ligation is complete, the ligated products can be released from crosslinked cytoskeleton cages. An overnight proteinase K digestion is used to degrade all the protein structures in the samples, releasing the chimeric Pore-C polymers into solution as dsDNA.

Day 3

On the final day of the protocol, the Pore-C DNA extract can be isolated. Following the overnight proteinase K digestion, the chimeric Pore-C dsDNA polymers are in a mixed solution of polypeptide fragments and residual reaction buffers. A phenol:chloroform extraction is used to remove the peptides from the sample, followed by an ethanol precipitation to purify the DNA from the residual reaction buffers and phenol.

The final product is the Pore-C DNA extract, which is a pool of chimeric dsDNA polymers made of multiple ligated monomers. These were originally in proximity within cells at the time of crosslinking at the start of the protocol. By sequencing the junctions between these monomers, inferences can be made about DNA interactions, proximity in sequence space, and the three-dimensional structures of chromatin within the nucleus.

The first steps of the protocol involve crosslinking chromatin within cells or nuclei. For mammalian cell culture suspensions, this process requires approximately 30 minutes of hands-on time over the course of the 80-minute experimental procedure. For other sample types, the process requires additional sample preparation steps which may increase the procedure time.

For any sample type, the crosslinking process is the only point at which the protocol may be paused. We recommend proceeding directly to DNA extraction or freezing samples at -80°C for subsequent use. Pausing the protocol in subsequent steps beyond this point is not recommended or supported. Subsequent extraction steps may not be delayed and must occur consecutively. Users are advised to ensure they have enough time to complete the protocol prior to starting extraction.

It is not advised to extend incubation times as they are unlikely to improve the quality of Pore-C data and may have a detrimental impact on the efficiency of de-crosslinking the DNA.

In total, for mammalian cell culture suspensions, this protocol requires approximately 100 minutes of hands-on time over the course of at least three days of experimental procedures including overnight incubations. Other sample types may require additional steps which may increase the time, but the full extraction can still be achieved within three days of experimental procedures.

4-cutter vs 6-cutter enzymes When selecting a restriction enzyme for the in situ digestion, bear in mind that the recognition site of the enzyme may impact the distribution of Pore-C contacts in the final Pore-C DNA extract. Restriction enzymes with six base recognition sites (6-cutters) will, on average, cut less frequently. Therefore, they will yield longer fragment monomers than restriction enzymes with four base recognition sites (4-cutters). Conversely, Pore-C extracts generated using 4-cutters will cut more frequently and may be expected to have higher contact densities compared to those generated using 6-cutters. Restriction enzymes with dual recognition sites, degenerate recognition sites, or recognition sites longer than six bases are more likely to produce biased cleavage distributions and are not recommended.

We recommend first testing a candidate restriction enzyme with an in silico restriction digestion to assess whether there are potential areas of reduced cleavage or repeat rich regions particularly susceptible to increased cleavage. If this is not possible due to poor genome reference quality, and you are unable to choose a restriction enzyme, then we strongly recommend proceeding with NlaIII as a default choice. Our investigations have found the NlaIII is particularly suitable for Pore-C extraction across many different species, yielding Pore-C extracts with high contact densities and desirable fragment lengths.

Heat denaturation vs chemical denaturation When choosing a restriction enzyme for this protocol, it important to note that chemical denaturation is required for restriction enzymes that denature at temperatures >65°C. Chemical denaturation causes a considerable reduction to the cis:trans ratio of Pore-C contacts (Nagano et al., 2015; Belaghzal, Dekker and Gibcus, 2017), therefore yield of useful contact data; increased trans-chromosomal contacts occur due to reduced integrity of nuclear structures. It is not advisable to attempt heat denaturation at temperatures >65°C as this may result in unintended crosslink reversal prior to proximity ligation, which will have a detrimental impact on the cis:trans ratio of Pore-C contacts (Belton et al., 2012). We highly recommend selecting an enzyme that does not require chemical denaturation and can be heat-denatured at temperatures <65°C.

Using multiple restriction enzymes Sample input requirements are recommended for optimal formaldehyde crosslinking. However, not all the crosslinked material is utilised for subsequent Pore-C extraction. In the case of mammalian cell suspensions, two pellets of ~5 million crosslinked cells are generated. Each cell pellet may be expected to have an initial yield of >7 μg Pore-C DNA extract, sufficient for SPRI size selection and sequencing library preparations. Different restriction enzymes will digest the genome with distinct cleavage distributions; users should consider the benefits of processing the two ~5 million crosslinked cell pellets separately using contrasting restriction enzymes (Kadota et al., 2019). This would serve to reduce biases encountered when using a single restriction enzyme for both cell pellets. However, this is an option that is at the discretion of the user and not essential.

The RE-Pore-C protocol has been developed to use a range of sample types as input, and some may require preparation following the relevant recommended Pore-C sample preparation procedure in the RE-Pore-C folder. There is also a separate protocol for plant Pore-C, which differs from the RE-Pore-C protocol to accommodate plant samples (see Figure 2). The Pore-C scaffolding of genomic assemblies: a case study of multiple sample types application note showcases how the Pore-C method can be applied to different sample types.

Figure 2. Pore-C processes required for different sample types. Most sample types may be processed through the RE-Pore-C protocol. However, plant samples require slightly different steps indicated in green for the plant Pore-C protocol.

Having read and considered the Protocol considerations section of this info sheet, proceed with the appropriate sample prep and extraction protocol. Where applicable, apply the options you have decided on having considered the information in this info sheet, such as the choice of restriction enzyme.

RE-Pore-C protocol

Restriction enzyme Pore-C protocol

Extraction of cell culture: ~10 million cells is recommended for input directly into the Re-Pore-C protocol:

• Cell suspensions: If the cell pellet is frozen, allow the pellet to thaw on ice. It is assumed frozen cells become compromised during freeze-thaw, which may result in over-estimation of viable cell count inputs prior to crosslinking. Despite this, we have found that frozen cell stocks may yield many micrograms of Pore-C DNA extract.

• Adherent cells: Cultures of adherent cells are recommended to be crosslinked whilst still adhered by adding the formaldehyde crosslinking solution directly to the culture flask. Volumes of crosslinking solution may be scaled up to ensure cells are submerged. Take note, this will require increased volumes of glycine for quenching.

  
  __Extraction of other sample preparations:__

• Peripheral blood mononucleocytes (PBMCs): a PBMC pellet harvested from ~5 ml of fresh whole blood is recommended.

• Animal tissue: ~50-100 mg of cryo-ground animal tissue is recommended.

• Insect material: ~50-100 mg of cryo-ground insect material is recommended.

• C. elegans material: ~1 ml of cryo-ground worm powder is recommended.

### Plant Pore-C protocol

• Plant Restriction enzyme Pore-C protocol: crosslinked nuclei isolated from ~2 g of plant material is recommended as input following the RE-Pore-C protocol.

Pore-C DNA extracts produced by following this protocol may be expected to yield an average fragment size of <10 Kbp when sequenced natively. Moreover, short reads of non-chimeric Pore-C monomers (or singletons) will not provide any contact information. To deplete non-chimeric monomers and maximise the frequency of chimeric Pore-C polymers we recommend a SPRI size selection, following the protocol as described but using a 0.85X custom SPRI bead ratio rather than the 0.7X described. If the initial yield Pore-C DNA extract is <1 μg and insufficient for SPRI size selection see below.

PCR amplification is not required for RE-Pore-C library prep as the Pore-C DNA extract can be sequenced natively. However, if a low yield of Pore-C DNA extract is obtained, either before or after size selection, where there is insufficient mass to proceed with a native ligation library preparation, it is possible to proceed with library preparation using a PCR-based approach (we recommend the Rapid PCR Barcoding Kit, SQK-RPB004). Libraries generated using the Rapid PCR Barcoding Kit are likely to generate more raw sequencing data compared with native (non-amplified) libraries, but tend to have an approximate number of contacts per run due to the shorter length of the amplicons generated. Therefore, the resulting data may be expected to have a higher cis:trans ratio.

We recommend the following approaches to preparing a sequencing library, according to the initial yield of Pore-C DNA extract:

>1 μg initial extraction yield of Pore-C DNA extract

• Proceed with SPRI size selection, following the input requirement as detailed in the size selection protocol. Expect a recovery of approximately 80% relative to the input mass. Analyse ~100 ng of size-selected Pore-C DNA extract on an Agilent Bioanlyser 12,000 bp chip for average fragment size. We have found Pore-C DNA extracts obtained using either DpnII or NlaIII in situ digestion yield average fragment lengths of approximately 7 kb following SPRI size selection.
• Proceed with a Ligation Sequencing Kit. We recommend the SQK-LSK114 kit for maximum sequencing accuracy and output. Older versions of the kit: SQK-LSK110 or SQK-LSK109 can also be used. Library preparation requires 100–200 fmols of SPRI size-selected Pore-C DNA extract as input. Follow the library preparation protocol as stated, however when it comes to the combined FFPE + Ultra II DNA repair and end-prep, incubate the reaction at 20°C for 15 minutes then 65°C for 5 minutes. Do not incubate at 20°C for 5 minutes then 65°C for 5 minutes as stated in the Ligation Sequencing Kit protocol. Before loading the flow cell, please see the advice on maximising throughout below.
• Lower molar inputs may be considered, however if SPRI size selection yields <40 fmols Pore-C DNA extract, proceed with a Rapid PCR Barcoding Kit (SQK-RPB004) library preparation.

500 ng–1 μg initial extraction yield of Pore-C DNA extract

• Insufficient mass of Pore-C DNA extract to carry out a SPRI size selection.
• Proceed with a Ligation Sequencing Kit. We recommend the SQK-LSK114 kit for maximum sequencing accuracy and output. Older versions of the kit: SQK-LSK110 or SQK-LSK109 can also be used. Library preparation requires 100–200 fmols of SPRI size-selected Pore-C DNA extract as input. Follow the library preparation protocol as stated, however when it comes to the combined FFPE + Ultra II DNA repair and end-prep, incubate the reaction at 20°C for 15 minutes then 65°C for 5 minutes. Do not incubate at 20°C for 5 minutes then 65°C for 5 minutes as stated in the Ligation Sequencing Kit protocol. Before loading the flow cell, see the advice on maximising throughout below.

100 ng–500 ng initial extraction yield of Pore-C DNA extract

• Proceed with a Rapid PCR Barcoding Kit (SQK-RPB004) library preparation following input requirements as detailed in the protocol.

Pore-C sequencing libraries made using the Ligation Sequencing Kit (SQK-LSK110 or SQK-LSK109) and sequenced on R9.4.1 flow cells may be expected to yield 1–2 Gb in 6 hours, or 4–8 Gb in 48 hours per flow cell on MinION Mk1B/GridION, as Pore-C DNA extracts are prone to blocking the pores. For this reason, we recommend initially loading flow cells with 20–40 fmols of sequencing library (double this for PromethION flow cells) and washing the flow cell using the Flow Cell Wash Kit (EXP-WSH004) to increase throughput.

Pore-C libraries made using the Ligation Sequencing Kit V14 (SQK-LSK114) and sequenced on R10.4.1 flow cells are expected to yield similar outputs per flow cell. Using this chemistry will yield greater resolution of repeat-rich sequences, which, in combination with the chromatin conformation contact mapping, would improve scaffolding of these regions in genomic assemblies (such as centromeres). Improved accuracy is beneficial to help scaffold "dark" regions of the genome such as the centromeres.

Over the course of the sequencing run, within approximately 18 hours, the number of available channels will likely decrease. When fewer than 20% of the channels remain available, we strongly recommend a flow cell wash to restore pore availability: this will require the Flow Cell Wash Kit (EXP-WSH004). Following the wash, reload the flow cell with another 20–40 fmols of sequencing library (double this for PromethION flow cells) - this will require the Sequencing Auxiliary Vials expansion (EXP-AUX001 if using SQK-LSK109, and EXP-AUX002 for SQK-LSK110 or EXP-AUX003 for SQK-LSK114). Repeating this process every time fewer than 20% of the channels remain available will optimise throughput.

The following kit requirements are based on several assumptions:

• a final library prep yield of >100 fmols
• a pore blocking profile that will require flow cell washes and re-loading of library within 18 hours or less
• a total run time of 72 hours

For a MinION Mk1B/GridION flow cell, you will need:

Kit	Number of kits needed per flow cell	Number of kits needed to maximise the use of one Ligation Sequencing Kit
Ligation Sequencing Kit	1	1
Flow Cell Wash Kit (EXP-WSH004)	1	6
Sequencing Auxiliary Vials expansion	1	1

For a PromethION flow cell, you will need:

Kit	Number of kits needed per flow cell	Number of kits needed to maximise the use of one Ligation Sequencing Kit
Ligation Sequencing Kit	1	1
Flow Cell Wash Kit (EXP-WSH004)	1	3
Sequencing Auxiliary Vials expansion	1	1

Note: flow cell washes are not required for sequencing libraries generated using a Rapid PCR Barcoding Kit (SQK-RPB004).

Sequence data generated from libraries constructed using Pore-C extracts can be analysed using the Pore-C workflow, which creates Hi-C outputs that can then be used for further analysis at the user's discretion.

For more information, please navigate to the GitHub repository and follow the instructions in the README file.

Oxford Nanopore Technologies has carried out Pore-C extractions on human cell line suspensions of NA12878, HG002/NA24385, and the cancer cell line HCC1954. We have primarily used the 4-cutters DpnII and NlaIII in our experiments.

Example read lengths of NlaIII- or DpnII-digested Pore-C extract sequencing data

Example throughput of Pore-C sequencing runs. Left: Library sequenced on a GridION with four flow cell washes. Right: Library sequenced on a PromethION with one flow cell wash. Throughput can be increased greatly with the use of flow cell washes. Please see the Library preparation section for more details.

Example Pore-C sequencing data metrics for NlaIII- or DpnII-digested Pore-C extracts.

Example Pore-C performance for NlaIII- or DpnII-digested Pore-C extracts.

We have released a Pore-C apps update regarding the impact of restriction enzymes on Pore-C performance and assemblies. Enzyme choice can impact the utility of Pore-C data and we have carried out four Pore-C DNA extractions in parallel using foud different restriction enzymes: the 4-cutters DpnII and NlaIII, along with the 6-cutters HindIII and SphI.

Deshpande, A.S., Ulahannan, N., Pendleton, M. et al. (2022) Identifying synergistic high-order 3D chromatin conformations from genome-scale nanopore concatemer sequencing. Nat Biotechnol. doi: 10.1038/s41587-022-01289-z

Belaghzal, H., Dekker, J. and Gibcus, J. H. (2017) Hi-C 2.0: an optimized Hi-C procedure for high-resolution genome-wide mapping of chromosome conformation. Methods, 123, pp. 56–65. doi: 10.1016/j.ymeth.2017.04.004.HI-C.

Belton, J.-M. et al. (2012) Hi-C: a comprehensive technique to capture the conformation of genomes. Methods, 58(3), pp. 1–16. doi: 10.1016/j.ymeth.2012.05.001.Hi-C

Comet, I. et al. (2011) A chromatin insulator driving three-dimensional Polycomb response element (PRE) contacts and Polycomb association with the chromatin fiber. Proceedings of the National Academy of Sciences, 108(6), pp. 2294–2299. doi: 10.1073/pnas.1002059108

Gavrilov, A. A., Golov, A. K. and Razin, S. V. (2013) Actual Ligation Frequencies in the Chromosome Conformation Capture Procedure. PLoS ONE, 8(3), pp. 1–6. doi: 10.1371/journal.pone.0060403

Kadota, M. et al. (2019) Multifaceted Hi-C benchmarking: what makes a difference in chromosome-scale genome scaffolding? bioRxiv, p. 659623. doi: 10.1101/659623.

Lieberman-Aiden, E. et al. (2009) Comprehensive mapping of long range interactions reveals folding principles of the human genome. Science, 326, pp. 289–293.

Nagano, T. et al. (2015) Comparison of Hi-C results using in-solution versus in-nucleus ligation. Genome Biology, 16(1), pp. 1–13. doi: 10.1186/s13059-015-0753-7

Sati, S. and Cavalli, G. (2017) Chromosome conformation capture technologies and their impact in understanding genome function. Chromosoma, 126(1), pp. 33–44. doi: 10.1007/s00412-016-0593-6

Ulahannan, N et al. (2019) Nanopore sequencing of DNA concatemers reveals higher-order features of chromatin structure. bioRxiv, p. 833590. doi: 10.1101/833590

25th January 2022: Pore-C scaffolding of genomic assemblies: a case study of multiple sample types

19th February 2021: Pore-C in situ restriction digestion and its impact on scaffolding genomic assemblies

9th October 2020: Assembly of Orzya sativa 'Azucena' rice genome at chromosome level resolution using long-read and Pore-C nanopore sequencing