TITLE: Flongle DirectRNA Library preparation
AUTHORS: Maximilian Krause

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Oxford Nanopore Technologies allows sequencing of native RNA for the first time. Additionally they released tiny devices that democratize sequencing among scientists. However, the smallest sequencing device - Flongle - is so far not officially supported for RNA sequencing experiments.</div><div class = "text-block"><span>This protocol provides users with a personally tested framework protocol to use Flongle flow-cells for native RNA sequencing. It is based on the protocol for </span><a href="https://community.nanoporetech.com/protocols/direct-rna-sequencing-sqk-rna002/v/DRS_9080_v2_revK_14Aug2019" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">ONT RNA library preparation</span></a><span> and changed to fit the volume and input requirements for Flongle.</span></div><div class = "text-block">The here advised workflow will routinely result in 100,000 native RNA reads within 24 hours of sequencing.</div></div>

[STEPS]
?. [Flongle native RNA library preparation (SQK-RNA002)]
Take  poly(A)-selected RNA into a 0.2ml thin-walled DNA-free PCR tube and bring volume to  with RNase-free water
200 ng
9.5 µl
The following description of Nanopore Library preparation is based on the protocols and consumable recommendations available at the date of publication (product version SQK-RNA002). However, experience has shown that Oxford Nanopore regularly updates protocols and the associated reagents to increase performance. Please check the current version of protocols at nanoporetech.com
If your RNA concentration is too low and upconcentration is necessary, use RNAClean XP bead procedures to increase the concentration of your RNA.
?. [Flongle native RNA library preparation (SQK-RNA002)]
Add the following reagents and carefully mix by pipetting: Nanopore RT adapter (RTA) of NEBNext Quick Ligation buffer T4 DNA Ligase ()
0.5 µl
3 µl
1.5 µl
[same as Quick T4 Ligase]
?. [Flongle native RNA library preparation (SQK-RNA002)]
(optionally) add  RNA CS from the Nanopore kit to monitor sequencing quality
0.5 µl
?. [Flongle native RNA library preparation (SQK-RNA002)]
Incubate for  at
25 Room temperature
Longer time can increase ligation efficiency, yet increase the chance of further RNA degradation
?. [Flongle native RNA library preparation (SQK-RNA002)]
Meanwhile, mix the following ingredients for a reverse-transcription Master Mix from SuperScript III kit: RNase-free water dNTPs First-Strand RT Buffer DTT
9 µl
2 µl
8 µl
4 µl
The following reverse transcription reaction is optional to remove secondary structures from RNA and increase RNA stability (in an RNA-DNA hybrid). Yet it may not be necessary for sequencing performance and can be omitted if wished. If these steps are omitted, the volume of RNAclean XP beads in step 60 have to be adjusted to 27ul
?. [Flongle native RNA library preparation (SQK-RNA002)]
After RNA incubation, add the Master Mix to the RNA sample and mix by careful pipetting
?. [Flongle native RNA library preparation (SQK-RNA002)]
Add  SuperScript III RT enzyme and mix by careful pipetting
2 µl
?. [Flongle native RNA library preparation (SQK-RNA002)]
In a thermocycler, incubate at  for ,  for  and finally bring to
50 °C
70 °C
4 °C
The incubation times can be reduced upon experience, as reverse transcription is optional and these incubation times are for most complete reverse transcription
?. [Flongle native RNA library preparation (SQK-RNA002)]
Transfer whole volume into a fresh Eppendorff 1.5ml Lo-Bind safe-lock tube
It is extremely important to work with the recommended DNA LoBind 1.5ml Eppendorff tubes. A series of experiments has shown that unknown plastic components from other tube do not only reduce the efficiency of DNA recovery, but also severely disturb the final sequencing chemistry, resulting in poor sequencing performance!
?. [Flongle native RNA library preparation (SQK-RNA002)]
Add  RNAClean XP beads and resuspend by careful pipetting
72 µl
?. [Flongle native RNA library preparation (SQK-RNA002)]
Incubate at  under constant agitation for
4 Room temperature
Every incubation step for purification in this protocol is slightly longer as recommended in Nanopore protocols. This is to increase efficiency of the reaction while at the same time minimizing RNA degradation. Shorter times might give more contiguous RNA reads at the expense of RNA quantity and thus library performance efficiency.
?. [Flongle native RNA library preparation (SQK-RNA002)]
Pellet beads on a magnetic stand and aspirate supernatant
?. [Flongle native RNA library preparation (SQK-RNA002)]
Wash the beads on the magnet with  fresh  EtOH without resuspending the beads. Instead, turn the tube quickly by 180°C to let the magnets float through the EtOH
200 µl
?. [Flongle native RNA library preparation (SQK-RNA002)]
Aspirate EtOH, spin down briefly on tabletop centrifuge and remove residual EtOH
?. [Flongle native RNA library preparation (SQK-RNA002)]
Resuspend beads with  RNase-free water by tapping the tube
10 µl
?. [Flongle native RNA library preparation (SQK-RNA002)]
Incubate  at
4 Room temperature
?. [Flongle native RNA library preparation (SQK-RNA002)]
Pellet beads on the magnetic stand and transfer to a new 1.5ml Lo-bind Safe-lock tube
?. [Flongle native RNA library preparation (SQK-RNA002)]
Add the following reagents for Sequencing adapter ligation: NEBNext Quick Ligation buffer Nanopore RNA Adapter Mix (RMX) RNase-free water T4 DNA Ligase  ()
4 µl
2 µl
2.5 µl
1.5 µl
[same as Quick T4 Ligase]
?. [Flongle native RNA library preparation (SQK-RNA002)]
Carefully mix by pipetting and incubate  at
4 Room temperature
?. [Flongle native RNA library preparation (SQK-RNA002)]
Add RNAClean XP beads and resuspend by careful pipetting
20 µl
?. [Flongle native RNA library preparation (SQK-RNA002)]
Incubate at  under constant agitation for
4 Room temperature
?. [Flongle native RNA library preparation (SQK-RNA002)]
Pellet beads on a magnetic stand and aspirate supernatant
?. [Flongle native RNA library preparation (SQK-RNA002)]
Wash the beads on the magnet with  Nanopore Wash Buffer (WSB) by resuspending the beads by tube-flicking
100 µl
?. [Flongle native RNA library preparation (SQK-RNA002)]
Aspirate Wash Buffer and repeat washing
?. [Flongle native RNA library preparation (SQK-RNA002)]
Aspirate Wash Buffer, spin down briefly on tabletop centrifuge and remove residual liquid
?. [Flongle native RNA library preparation (SQK-RNA002)]
Resuspend beads with  Elution buffer water by tapping the tube
9 µl
?. [Flongle native RNA library preparation (SQK-RNA002)]
Incubate  at
4 Room temperature
?. [Flongle native RNA library preparation (SQK-RNA002)]
Pellet beads on the magnetic stand and transfer to a new 1.5ml Lo-bind Safe-lock tube
?. [Flongle native RNA library preparation (SQK-RNA002)]
Use  to quantify final library on Qubit DNA HS Kit
1 µl
Use the RNA HS kit if you omitted the cDNA synthesis, as the DNA kit is sensitive to double-stranded nucleotide sequences only. Recovery aim is around 80ng
?. [Flongle native RNA library preparation (SQK-RNA002)]
To the recovered  RNA library, add  RNase-free water and  Nanopore Sequencing Buffer (RRB), mix by careful pipetting
8 µl
7 µl
15 µl
?. [Flongle native RNA library preparation (SQK-RNA002)]
Prime the Flongle Flow cell with the  Flush Buffer mix (a detailed description including video documentation can be found here)
120 µl
Most important during Priming and loading is to not use any force when applying reagents, and to avoid introduction of air bubbles. Both physical force and air bubble introduction can rupture sequencing arrays and clog essential microfluidic valves, which make later use of flow cells impossible.
If unsure, dispense volume by turning the adjustment wheel of your pipet instead of dispensing as usual
?. [Flongle native RNA library preparation (SQK-RNA002)]
Start sequencing on MinKNOW (at least version 19.12.5) by choosing Flongle as Flow-cell and RNA-002 as kit. (Other options depending on preference for data output; external Link: Start Sequencing)
?. [Flongle native RNA library preparation (SQK-RNA002)]
Use the time to quality-control the Flongle Flow Cell (Insert Flongle adapter and Flow Cell in the MinION sequencer, and run the "Test Flow Cell" program in MinKNOW).
?. [Flongle native RNA library preparation (SQK-RNA002)]
Mix  Flush Buffer (FLB) and  Flush Tether (FLT) in a new 1.5ml Lo-bind Safe-lock tube
117 µl
3 µl
?. [Flongle native RNA library preparation (SQK-RNA002)]
Immediately after priming, add the 30ul Library mix carefully in the Flongle Flow Cell
If unsure, dispense volume by turning the adjustment wheel of your pipet instead of dispensing as usual