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Extraction time
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.
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Choosing a restriction enzyme
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.