The QIAmp Viral RNA Kit was used to isolate RNA out of viral supernatant following the manufacture's protocol.
• 560 µl prepared Buffer AVL (containing 5.6 µg carrier RNA)
• 140 µl cell-culture supernatant
• Incubate 10 min
• 560 µl ethanol (100%)
• 500 µl Buffer AW1
• 500 µl Buffer AW2
• Elute in 60 µl Buffer AVE
Thanks to Mathias Schemmerer for providing HepE infected cell cultures.
The RNA amplification was performed with the following approach and PCR protocol for every single amplicon.
PCR reaction:
• 12,5 μL 2X Platinum™ SuperFi™ RT-PCR Master Mix
• 0.25 μL SuperScript™ IV RT Mix
• 0.75 µl Nuclease-free water
• 9.00 µl Template RNA (0.01 pg to 1 μg total RNA)
• 1.25 μl varVAMP´s forward primer (10 μM)
• 1.25 μl varVAMP´s reverse primer (10 μM)
Total volume: 25 µl
PCR cycler program (Touchdown):
| Mode | Output | Description |
|---|---|---|
| 55°C | 60 min | 1 |
| 98°C | 2 min | 1 |
| 98°C | 10s | 10 |
| 63°C (-0.5°C/cycle) | 10s | 10 |
| 72°C | 2 min | 10 |
| 98°C | 10s | 35 |
| 58°C | 10s | 35 |
| 72°C | 2 min | 35 |
| 72°C | 5 min | 1 |
| 4°C | ∞ | 1 |
INFO: Primers can also be used in two multiplex reactions.
Figure 1: The gel electrophoresis (1% agarose gel) indicated bands for all the single amplicons in the equivalent amplicon size of the design and no band for the negative control (NTC).
After pooling the single PCR products of the seven amplicons the library preparation was performed with the NEBNext® Ultra™ II FS DNA Library Prep Kit for Illumina®. Next generation sequencing was performed with an Ilumina MiSeq (2x150 bp).
IMPORTANT: This protocol was established by Johanna Antonia Kleine as part of her M.D. thesis. Many thanks for your great work!