Influenza A RNA
Extraction Method
Influenza A RNA
FOR RESEARCH USE ONLY
Contents
Materials
Methods
Results
Sequencing performance
Keller, M.W. et al., Direct RNA Sequencing of the Coding Complete Influenza A Virus Genome. Nature Scientific Reports, 8, 14408
Materials
- 200–2000 µl MDCK cell culture or embryonated chicken egg allantoic fluid
- TRIzol (Invitrogen)
- Phase Lock GelTM tubes (VWR) - optional
- RNase-free glycogen or GlycoBlue™ Coprecipitant (ThermoFisher)- optional
- 75% freshly-prepared ethanol
- Isopropanol
- Nuclease-free water or TE buffer
- 1.5 ml Eppendorf DNA LoBind tubes
- 1.5 ml Eppendorf tubes
- Custom Reverse Transcription Adapter (RTA) for library prep (see below)
Methods
Critical Step: Ensure you start with at least 3X more TRIzol than the volume of your starting material. Extract 250 µl of sample in a 1.5 ml Eppendorf DNA LoBind tube. For example, if you have 1000 µl starting material, four tubes will be used.
Follow the standard TRIzol protocol, using a Phase Lock GelTM tube to trap the organic phase. Add 1 µg RNase-free glycogen to the first isopropanol precipitation. Then use standard 1.5 ml tubes for the pelleting steps.
Optional Step: GlycoBlue Coprecipitant can be used to make the pellet visible.
Critical Step: If the extractions were performed in multiple tubes, when resuspending the pellet in ethanol, use the same 1 ml of ethanol to serially resuspend all the pellets.
Elute the RNA in 10–100 µl nuclease-free water or TE buffer.
Results
- Yield: 10-1100 ng
Sequencing performance
Libraries were prepared using the Direct RNA Sequencing Kit with a custom RTA:
| Name | Sequence |
|---|---|
| RTA-A | /5phos/GGCTTCTTCTTGCTCTTAGGTAGTAGGTTC |
| RTA-B | GAGGCGAGCGGTCAATTTTCCTAAGAGCAAGAAGAAGCCTTTTTTTTTT |
| RTA-B-U12 | GAGGCGAGCGGTCAATTTTCCTAAGAGCAAGAAGAAGCCAGCAAAAGCAGG |
| RTA-A-U12.4 | GAGGCGAGCGGTCAATTTTCCTAAGAGCAAGAAGAAGCCAGCGAAAGCAGG |
The custom RTAs can be purchased from IDT, with each of the modified RTA-B strands already duplexed to the RTA-A strand. The RTA-A has a 5’ phosphate modification for ligation. The regions of reverse complementarity between the RTA strands are underlined, and the target sequences are coloured.
- Read length profile:
