Keller, M.W. et al., Direct RNA Sequencing of the Coding Complete Influenza A Virus Genome. Nature Scientific Reports, 8, 14408

• 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)

1. 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.

2. 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.

3. Optional Step: GlycoBlue Coprecipitant can be used to make the pellet visible.

4. 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.

5. Elute the RNA in 10–100 µl nuclease-free water or TE buffer.

• Yield: 10-1100 ng

Libraries were prepared using the Direct RNA Sequencing Kit with a custom RTA:

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: