Feb 24, 2020
Flongle DirectRNA Library preparation
- 1University of Bergen
- The Nordic Nanopore-Seq Network
External link: http://doi:10.1261/rna.071332.119
Protocol Citation: Maximilian Krause 2020. Flongle DirectRNA Library preparation. protocols.io https://dx.doi.org/10.17504/protocols.io.bcwcixaw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
This protocol was used on four different occasions and regularly produced >80,000 reads.
Created: February 24, 2020
Last Modified: February 24, 2020
Protocol Integer ID: 33444
Keywords: Nanopore, Direct RNA Sequencing, Flongle, RNA-Seq
Abstract
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.
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 ONT RNA library preparation and changed to fit the volume and input requirements for Flongle.
The here advised workflow will routinely result in 100,000 native RNA reads within 24 hours of sequencing.
Guidelines
One of the main considerations to take for any Nanopore sequencing experiment is that read length affects output quality and quantity. Therefore EVERY experimental step should be reviewed for forces that could generate molecule degradation. Thus we advise against any vortexing and forceful pipetting during the following procedures. Instead, we advise to handle samples with care and mix by tube inversion wherever possible. Keeping samples on ice is not recommended, as it could reduce ligation efficiencies, but could be considered for any short pausing steps.
The actual Library preparation protocol has NO safe stopping point. Thus please make sure you have sufficient time for the final steps of the library.
Oxford Nanopore library preparation is based on the ligation of a bridge adapter specific to the poly(A) tail, and the subsequent addition of a Motor Protein adapter based on sequence complementarity to the first adapter. The efficiency of library preparation thus solely depends on the efficiency of DNA-RNA ligation procedures. Any contaminant that reduces ligation efficiency will impact the final library performance.
Additionally, any RNA species without poly(A) tails that could interfere with the ligation (unspecific binding) have an effect on ligation efficiency. It is thus important to follow the recommendations given in the Nanopore protocols (nanoporetech.com) for RNA quality and quantity measures.
Finally, it is crucial to proceed quickly from the final ligation to actual sequencing and avoid harsh chemicals and temperatures with that library, as an active protein is added whose function is essential for sequencing.
Materials
MATERIALS
NEBNext Quick Ligation Module - 20 rxnsNew England BiolabsCatalog #E6056S
Qubit dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q32851
Agencourt RNAClean XP BeadsBeckman CoulterCatalog #A63987
PCR Machine
95% EtOH
Nuclease-free waterThermo Fisher ScientificCatalog #R0581
Centrifuge 5424 R refrigerated with Rotor FA-45-24-11 rotary knobs 120 V/50 – 60 Hz (US)EppendorfCatalog #5404000537
Qubit FluorometerLife TechnologiesCatalog #Q33216
DNA LoBind Tubes, 1.5 mLEppendorfCatalog #0030108051
Qubit RNA HS Assay KitThermo Fisher ScientificCatalog #Q32852
Qubit assay tubesThermo Fisher ScientificCatalog #Q32856
DynaMag™-2 MagnetThermo FisherCatalog #12321D
HulaMixer™ Sample MixerThermo FisherCatalog #15920D
SuperScript™ III Reverse TranscriptaseThermo FisherCatalog #18080044
Thin-walled, frosted lid, RNase-free PCR tubes (0.2 mL)Thermo FisherCatalog #AM12225
dNTP Mix (10 mM each)Thermo FisherCatalog #R0191
Direct RNA Sequencing kit (SQK-RNA002)Oxford Nanopore TechnologiesCatalog #SQK-RNA002
Flow Cell Priming Kit (EXP-FLP002)Oxford Nanopore TechnologiesCatalog #EXP-FLP002
MinION sequencerOxford Nanopore Technologies
ONT Flongle Flow-CellOxford Nanopore TechnologiesCatalog #FLO-FLG001
ONT Flongle adapterOxford Nanopore TechnologiesCatalog #FLGIntSP
The specific enzymes recommended for use in the library preparation are under constant review by Oxford Nanopore Technologies. Please visit the company's website and protocols for possible updates on performance-enhancing chemistry.
The Flow Cell Priming Kit (EXP-FLP002 in this instance) is usually a component of the Library preparation kit and does not have to be ordered extra.
Before start
This protocol is based on the "Direct RNA sequencing (SQK-RNA002)" protocol from Oxford Nanopore Technologies. The protocol is available for Community members here.
Please check for updates on these protocols, and check your RNA kit availability, as the kit chemistry develops fast. A new version of this kit (SQK-RNA003) is already inbuilt in the recent MinKNOW update. However, the comments and recommendations for basic incubation steps in this protocol will be valid for upcoming versions as well.
RNA should be extracted as fresh as possible, or alternatively stored at -80°C in RNA storage medium (TRI reagent or RNALater). The sample size should be chosen big enough to yield the required amount of poly(A)-selected RNA - currently 200ng. As mRNA is routinely only 1-2% of total RNA, it should be aimed for extracting 10ug of total RNA from the sample. A full workflow including RNA extraction and poly(A) enrichment can be found here.
General idea
- Volumes for RTA and RMX were reduced to fit the Flongle input requirements (1/2 and 1/3 respectively.
- The volumes for the initial RTA ligation and Reverse transcription were not changed, to avoid pipetting of too small volumes.
- The volume of the RMX adapter ligation was reduced to 1/2 to account for smaller amount of RNA and save reagent costs.
- The volumes of the final library mix was adjusted to fit the volumes recommended in other Flongle sequencing protocols.
Flongle native RNA library preparation (SQK-RNA002)
Flongle native RNA library preparation (SQK-RNA002)
Take 200 ng poly(A)-selected RNA into a 0.2ml thin-walled DNA-free PCR tube and bring volume to 9.5 µL with RNase-free water
Note
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
Note
If your RNA concentration is too low and upconcentration is necessary, use RNAClean XP bead procedures to increase the concentration of your RNA.
Add the following reagents and carefully mix by pipetting:
- 0.5 µL Nanopore RT adapter (RTA)
- 3 µL of NEBNext Quick Ligation buffer
- 1.5 µL T4 DNA Ligase (2000 U/ul same as Quick T4 Ligase )
(optionally) add 0.5 µL RNA CS from the Nanopore kit to monitor sequencing quality
Incubate for 00:15:00 at Room temperature
Note
Longer time can increase ligation efficiency, yet increase the chance of further RNA degradation
15m
Meanwhile, mix the following ingredients for a reverse-transcription Master Mix from SuperScript III kit:
- 9 µL RNase-free water
- 2 µL 10 Molarity (M) dNTPs
- 8 µL First-Strand RT Buffer
- 4 µL 0.1 Molarity (M) DTT
Note
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
After RNA incubation, add the Master Mix to the RNA sample and mix by careful pipetting
Add 2 µL SuperScript III RT enzyme and mix by careful pipetting
In a thermocycler, incubate at 50 °C for 00:50:00 , 70 °C for 00:10:00 and finally bring to 4 °C
Note
The incubation times can be reduced upon experience, as reverse transcription is optional and these incubation times are for most complete reverse transcription
1h
Transfer whole volume into a fresh Eppendorff 1.5ml Lo-Bind safe-lock tube
Note
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!
Add 72 µL RNAClean XP beads and resuspend by careful pipetting
Incubate at Room temperature under constant agitation for 00:10:00
Note
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.
10m
Pellet beads on a magnetic stand and aspirate supernatant
Wash the beads on the magnet with 200 µL fresh 70 % volume EtOH without resuspending the beads. Instead, turn the tube quickly by 180°C to let the magnets float through the EtOH
Aspirate EtOH, spin down briefly on tabletop centrifuge and remove residual EtOH
Resuspend beads with 10 µL RNase-free water by tapping the tube
Incubate 00:10:00 at Room temperature
10m
Pellet beads on the magnetic stand and transfer to a new 1.5ml Lo-bind Safe-lock tube
Add the following reagents for Sequencing adapter ligation:
- 4 µL NEBNext Quick Ligation buffer
- 2 µL Nanopore RNA Adapter Mix (RMX)
- 2.5 µL RNase-free water
- 1.5 µL T4 DNA Ligase (2000 U/ul same as Quick T4 Ligase )
Carefully mix by pipetting and incubate 00:15:00 at Room temperature
15m
Add 20 µL RNAClean XP beads and resuspend by careful pipetting
Incubate at Room temperature under constant agitation for 00:10:00
10m
Pellet beads on a magnetic stand and aspirate supernatant
Wash the beads on the magnet with 100 µL Nanopore Wash Buffer (WSB) by resuspending the beads by tube-flicking
Aspirate Wash Buffer and repeat washing go to step #23
Aspirate Wash Buffer, spin down briefly on tabletop centrifuge and remove residual liquid
Resuspend beads with 9 µL Elution buffer water by tapping the tube
Incubate 00:10:00 at Room temperature
10m
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).
Pellet beads on the magnetic stand and transfer to a new 1.5ml Lo-bind Safe-lock tube
Use 1 µL to quantify final library on Qubit DNA HS Kit
Note
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
To the recovered 8 µL RNA library, add 7 µL RNase-free water and 15 µL Nanopore Sequencing Buffer (RRB), mix by careful pipetting
Mix 117 µL Flush Buffer (FLB) and 3 µL Flush Tether (FLT) in a new 1.5ml Lo-bind Safe-lock tube
Prime the Flongle Flow cell with the 120 µL Flush Buffer mix (a detailed description including video documentation can be found here)
Note
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.
Note
If unsure, dispense volume by turning the adjustment wheel of your pipet instead of dispensing as usual
Immediately after priming, add the 30ul Library mix carefully in the Flongle Flow Cell
Note
If unsure, dispense volume by turning the adjustment wheel of your pipet instead of dispensing as usual