This protocol describes the preparation of Rattus norvegicus (R. norvegicus) tissue samples to be processed using the restriction enzyme Pore-C (RE-Pore-C) protocol as an example of animal tissue. This protocol was developed using brain and muscle tissues isolated from the Hsd:Sprague Dawley (SD) strain of Rattus norvegicus domestica subspecies.

• 100 mg tissue (e.g. brain or muscle)
• 1X PBS pH 7.4
• Crushed ice
• Liquid nitrogen
• Scalpel
• 15 or 50 ml centrifuge tubes
• Mortar and pestle
• –80°C freezer storage

Note: Pre-chill a mortar and pestle at –80°C for at least 30 minutes. Both fresh or frozen samples may be used.

1. Gather 100 mg of animal tissue. Note: If the material is >1 cm2, dissect the sample into smaller pieces before proceeding to the next step.

2. Place a chilled mortar and pestle on ice. Add a small volume of liquid nitrogen into the mortar and add the animal tissue to freeze until the liquid nitrogen has evaporated.

3. Carefully grind the frozen animal tissue into a fine powder, working quickly to minimise thawing. If the animal tissue starts to thaw, add a small volume of liquid nitrogen to the mortar.

4. Use a spatula to collect the tissue powder into a chilled centrifuge tube on ice

1. Transfer approximately 100 mg of cryo-ground tissue to a 50 ml centrifuge tube and resuspend in 1 ml chilled 1X PBS.

2. Bring the volume of the re-suspended cryo-ground tissue to 10 ml in chilled 1X PBS.

3. Proceed with the RE-Pore-C protocol, using the re-suspended cryo-ground tissue powder as input.

Table 1. The yield of non-size selected RE-Pore-C DNA extract using NlaIII restriction enzyme.

Figure 1. Agilent Bioanalyser DNA 12000 trace of non-size selected RE-Pore-C DNA extract.

Figure 2. The sequencing and Pore-C output for libraries assessed on PromethION. Libraries were generated as described using Pore-C extracts prepared with the NlaIII restriction enzyme. The read length distributions and output (Gbases) obtained from the libraries generated are shown in panels A and B, respectively. Panel C displays the Pore-C metrics obtained.