Introduction
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.
Materials
- 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
Cryo-grinding of animal tissues: 20 minutes hands-on-time
Note: Pre-chill a mortar and pestle at –80°C for at least 30 minutes. Both fresh or frozen samples may be used.
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.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.
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.
Use a spatula to collect the tissue powder into a chilled centrifuge tube on ice
RE-Pore-C extraction
Transfer approximately 100 mg of cryo-ground tissue to a 50 ml centrifuge tube and resuspend in 1 ml chilled 1X PBS.
Bring the volume of the re-suspended cryo-ground tissue to 10 ml in chilled 1X PBS.
Proceed with the RE-Pore-C protocol, using the re-suspended cryo-ground tissue powder as input.
Results
Sample | DNA concentration, ng/μl | Total DNA mass, μg |
---|---|---|
Muscle | 79.4 | 1.19 |
Brain | 14.9 | 2.24 |
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.
Change log
Version | Change |
---|---|
v2, 28th July 2020 | Title update |
v1, 20th July 2020 | Intial protocol publication |