Animal tissue preparation for RE-Pore-C
Oxford Nanopore
Animal tissue preparation for RE-Pore-C
FOR RESEARCH USE ONLY
Contents
Introduction
Materials
Results
Change log
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 |