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Sample types
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. -
Extraction protocols
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.