TITLE: ONT DirectRNA Library preparation for poly(A) estimation
AUTHORS: Maximilian Krause, Adnan M Niazi

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol provides a detailed explanation of of the steps necessary for successful Direct RNA Library preparation for Oxford Nanopore Sequencing. The protocol explains the steps needed for RNA sample preparation based on TRIzol extraction and Poly(A)Purist Mag kit enrichment prior to Direct RNA library preparation protocol. The library preparation protocol is based on the Library preparation protocols for RNA-002 kits, yet offers additional advice on what we think is important for a successful library with minimal RNA degradation.</div><div class = "text-block"><span>The protocol is used to assess poly(A) tail length using the </span><span style = "font-style:italic;">tailfindr</span><span> package. The poly(A) tail is a homopolymeric stretch of adenosine at the 3`-end of mature RNA transcripts and its length plays an important role in nuclear export, stability, and translational regulation of mRNA. With the introduction of native RNA sequencing by Oxford Nanopore Technologies (ONT), it is now possible to sequence full-length native RNA. A single long read contains both the transcript and the associated poly(A) tail, thereby making genome-wide transcript-specific poly(A) tail length assessment in native RNA feasible. For more information on </span><span style = "font-style:italic;">tailfindr</span><span> visit the </span><a href="https://rnajournal.cshlp.org/content/25/10/1229.abstract" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">publication</span></a><span> or </span><a href="https://github.com/adnaniazi/tailfindr" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">the GitHub repository</span></a></div></div>

[STEPS]
?. [RNA extraction and quality control]
Resuspend and homogenize necessary amount of fresh sample in TRIZol reagent (1ml of TRIZol per 50mg tissue or 3x10^7 cells) in an Eppendorf Safe-Lock 1.5ml tube
Homogenization should be kept as gentle as possible to avoid RNA molecule degradation. Reduce number of pestle strokes, pipetting, or replace by vigorous shaking.
?. [RNA extraction and quality control]
Incubate  at , with regular tube inversion
0 Room temperature
?. [RNA extraction and quality control]
Add  chloroform per TRIZOL and shake by tube inversion
200 µl
1 ml
?. [RNA extraction and quality control]
Incubate  at , with regular tube inversion
25 Room temperature
?. [RNA extraction and quality control]
Centrifuge  at 12-15,000g at  to separate phases
4 °C
?. [RNA extraction and quality control]
Carefully transfer the aqueous phase to a new Eppendorf Safe-lock 1.5ml tube by angling the tube for most efficient transfer
Care should be taken to avoid any transfer of TRI reagent. A small drop of aqueous phase can be left behind to make sure that the sample is as clean as possible.
?. [RNA extraction and quality control]
Add  GlycoBlue reagent, NaOAc and Isopropanol and mix by inversion of the tubeBy experience, samples with an initial volume of 1ml TRI reagent will need  NaOAc and Isopropanol
1 µl
0.1
1
50 µl
500 µl
?. [RNA extraction and quality control]
Incubate  at  for most efficient yields
-20 °C
?. [RNA extraction and quality control]
Centrifuge for  at 15-20,000g at
4 °C
Total RNA should form a strong white pellet. Care should be taken to not aspirate the pellet during the following washing steps
?. [RNA extraction and quality control]
Aspirate the supernatant without disturbing the RNA pellet
?. [RNA extraction and quality control]
Wash the RNA pellet with  freshly-prepared  EtOH
1 ml
?. [RNA extraction and quality control]
Centrifuge for  at 15-20,000g at
4 °C
?. [RNA extraction and quality control]
Aspirate supernatant and repeat ethanol wash  once
?. [RNA extraction and quality control]
Aspirate the supernatant and air-dry the pellet for
[can be reduced to 2 min]
If necessary, briefly spin down on a tabletop centrifuge to collect remaining EtOH, and pipet off with a 200ul pipet
?. [RNA extraction and quality control]
Add  of RNase-free water and resuspend by tapping the tube or shaking in a thermoshaker at
50 µl
25 Room temperature
?. [RNA extraction and quality control]
Record quantity and quality by Nanodrop measurement and Qubit RNA Broad Range kit. Test RNA integrity by BioAnalyzer RNA chip
All measurements are necessary for Nanopore Experiments. Nanodrop 260/280 and 260/230 measurements are important to assess possible remnant contaminants that are detrimental to Nanopore's sequencing chemistry. Qubit measurements offer the most sensitive RNA quantification, and are regularly used during the library preparation protocols. BioAnalyzer traces yield an RNA integrity measurement that allows to assess the biological quality of the sample
?. [poly(A) enrichment]
Bring RNA concentration to , but minimal  ( RNA)
50 µl
30 µg
Only consider total RNA samples with BioAnalyzer RIN (RNA Integrity number) higher than 9 for further procedures as RNA quality directly affects sequencing quality and quantity
?. [poly(A) enrichment]
Add an equal volume of the Poly(A)Purist Mag Kit 2x Binding Solution (minimal) and mix by tube inversion
50 µl
?. [poly(A) enrichment]
Store RNA  until further processing
on ice
?. [poly(A) enrichment]
Vortex the Poly(A)Purist Magnetic Bead solution and pipet the necessary amount of beads to a 1.9ml tube provided with the kit. For each  of total RNA from above, use  Magnetic Beads solution and in subsequent washing steps  Wash Buffer
100 µg
10 µl
50 µl
Never use smaller volumes than 10 ul beads and 50 ul Wash Buffer, as it will reduce efficiency of washes and RNA elution during the protocol
?. [poly(A) enrichment]
Precipitate the beads on a magnetic stand and aspirate the buffer
Beads might take several minutes to fully precipitate. Observe the buffer to check for clarity. Occasional slow rotation of the tubes on the magnetic stand may increase the collection efficiency
?. [poly(A) enrichment]
Take the tube out of the magnetic stand and resuspend the beads in Kit Wash Solution 1 with volume depending on the amount of magnetic beads used. For example,
?. [poly(A) enrichment]
Repeat once from
?. [poly(A) enrichment]
Add the total RNA sample + Binding Solution from   to the beads, mix by tube inversion
?. [poly(A) enrichment]
Heat the bead-RNA mixture to for
65 °C
Longer time and higher temperatures are not advised, to avoid additional RNA degradation
?. [poly(A) enrichment]
Incubate  at  under constant agitation
65 Room temperature
Longer incubation time to up to 1h is possible, but increases the chance of RNA degradation
?. [poly(A) enrichment]
Meanwhile preheat the Kit THE elution buffer to
70 °C
?. [poly(A) enrichment]
Precipitate the magnetic beads with the RNA attached on the magnetic stand and aspirate supernatant
?. [poly(A) enrichment]
Take the tube out of the magnetic stand and resuspend the beads in Kit Wash Solution 1 with volume depending on
?. [poly(A) enrichment]
Repeat once from
?. [poly(A) enrichment]
Take the tube out of the magnetic stand and resuspend the beads in Kit Wash Solution 2 with volume depending on
?. [poly(A) enrichment]
Precipitate beads on the magnetic stand and aspirate the supernatant
?. [poly(A) enrichment]
Repeat o
?. [poly(A) enrichment]
Briefly spin down on a tabletop centrifuge to remove residual Wash Solution
?. [poly(A) enrichment]
Remove the tube from the magnetic stand and resuspend in  hot THE buffer from
100 µl
?. [poly(A) enrichment]
Incubate  at
[can be extended to 2 min]
70 °C
This additional incubation is to make sure that elution efficiency is as high as possible. However, heat treatment of RNA should be kept short to avoid RNA degradation.
?. [poly(A) enrichment]
Capture the magnetic beads on the magnetic stand, and transfer the supernatant into a clean Eppendorf 1.5ml Safe-lock tube and store on ice
?. [poly(A) enrichment]
Repeat from   and pool the supernatants into one tube
?. [poly(A) enrichment]
Put the fresh sample tube on a magnet again to collect residual beads for
on ice
?. [poly(A) enrichment]
Transfer the cleaned supernatant into a fresh Eppendorf 1.5ml tube
?. [poly(A) enrichment]
Add GlycoBlue reagent,  NaAc and  Isopropanol, mix by inversion
1 µl
20 µl
250 µl
?. [poly(A) enrichment]
Store  at
-20 °C
?. [poly(A) enrichment]
Centrifuge for  at 15-20,000g at
4 °C
poly(A)-selected RNA should form a small white pellet, with blue coloring from the coprecipitant. Care should be taken to not aspirate the pellet during the following washing steps. If a small brown coloring is observed, it is residual magnetic beads that should not affect downstream processes.
?. [poly(A) enrichment]
Aspirate the supernatant without disturbing the RNA pellet
?. [poly(A) enrichment]
Wash the RNA pellet with  freshly-prepared  EtOH
1 ml
?. [poly(A) enrichment]
Centrifuge for  at 15-20,000g at
4 °C
?. [poly(A) enrichment]
Repeat once from
?. [poly(A) enrichment]
Aspirate the supernatant and air-dry the pellet for
If necessary, briefly spin down on a tabletop centrifuge to collect remaining EtOH, and pipet off with a 200ul pipet
?. [poly(A) enrichment]
Add  of RNase-free water and resuspend by tapping the tube or shaking in a thermoshaker at .
15 µl
25 °C
?. [poly(A) enrichment]
Record quantity and quality by Nanodrop measurement and Qubit RNA Broad Range kit. Test rRNA removal by BioAnalyzer RNA chip.
All measurements are necessary for Nanopore Experiments. Nanodrop 260/280 and 260/230 measurements are important to assess possible remnant contaminants that are detrimental to Nanopores sequencing chemistry. Qubit measurements offer the most sensitive RNA quantification, and are regularly used during the library preparation protocols. BioAnalyzer traces at this step will provide an estimate for effective rRNA removal. RIN numbers should be low, as RIN is calculated based on rRNA peaks. The length distribution should give an estimate of which average read length can be expected from Nanopore sequencing.
?. [Nanopore Direct 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
500 ng
9 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.
?. [Nanopore Direct 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 ()
1 µl
3 µl
1.5 µl
[same as Quick T4 Ligase]
?. [Nanopore Direct RNA library preparation (SQK-RNA002)]
(optionally) add  RNA CS from the Nanopore kit to monitor sequencing quality
0.5 µl
?. [Nanopore Direct 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
?. [Nanopore Direct 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
?. [Nanopore Direct RNA library preparation (SQK-RNA002)]
After RNA incubation, add the Master Mix to the RNA sample and mix by careful pipetting
?. [Nanopore Direct RNA library preparation (SQK-RNA002)]
Add  SuperScript III RT enzyme and mix by careful pipetting
2 µl
?. [Nanopore Direct 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
?. [Nanopore Direct 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!
?. [Nanopore Direct RNA library preparation (SQK-RNA002)]
Add  RNAClean XP beads and resuspend by careful pipetting
72 µl
?. [Nanopore Direct 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.
?. [Nanopore Direct RNA library preparation (SQK-RNA002)]
Pellet beads on a magnetic stand and aspirate supernatant
?. [Nanopore Direct 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
?. [Nanopore Direct RNA library preparation (SQK-RNA002)]
Aspirate EtOH, spin down briefly on tabletop centrifuge and remove residual EtOH
?. [Nanopore Direct RNA library preparation (SQK-RNA002)]
Resuspend beads with  RNase-free water by tapping the tube
20 µl
?. [Nanopore Direct RNA library preparation (SQK-RNA002)]
Incubate  at
4 Room temperature
?. [Nanopore Direct RNA library preparation (SQK-RNA002)]
Pellet beads on the magnetic stand and transfer to a new 1.5ml Lo-bind Safe-lock tube
?. [Nanopore Direct 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  ()
8 µl
6 µl
3 µl
3 µl
[same as Quick T4 Ligase]
?. [Nanopore Direct RNA library preparation (SQK-RNA002)]
Carefully mix by pipetting and incubate  at
4 Room temperature
?. [Nanopore Direct RNA library preparation (SQK-RNA002)]
Add RNAClean XP beads and resuspend by careful pipetting
40 µl
?. [Nanopore Direct RNA library preparation (SQK-RNA002)]
Incubate at  under constant agitation for
4 Room temperature
?. [Nanopore Direct RNA library preparation (SQK-RNA002)]
Pellet beads on a magnetic stand and aspirate supernatant
?. [Nanopore Direct RNA library preparation (SQK-RNA002)]
Wash the beads on the magnet with  Nanopore Wash Buffer (WSB) by resuspending the beads by tube-flicking
150 µl
?. [Nanopore Direct RNA library preparation (SQK-RNA002)]
Aspirate Wash Buffer and repeat washing
?. [Nanopore Direct RNA library preparation (SQK-RNA002)]
Aspirate Wash Buffer, spin down briefly on tabletop centrifuge and remove residual liquid
?. [Nanopore Direct RNA library preparation (SQK-RNA002)]
Resuspend beads with  Elution buffer water by tapping the tube
21 µl
?. [Nanopore Direct RNA library preparation (SQK-RNA002)]
Incubate  at
4 Room temperature
?. [Nanopore Direct RNA library preparation (SQK-RNA002)]
Pellet beads on the magnetic stand and transfer to a new 1.5ml Lo-bind Safe-lock tube
?. [Nanopore Direct 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
?. [Nanopore Direct RNA library preparation (SQK-RNA002)]
Add  RNase-free water and  Nanopore Sequencing Buffer (RRB) to the library
17.5 µl
37.5 µl
?. [Nanopore Direct RNA library preparation (SQK-RNA002)]
Prime a MinION flow cell as specified in Nanopore protocols, and finally load the library drop-wise through the Sample port (a detailed description including video documentation can be found here: Flow Cell Priming)
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.
Library loading by drop-wise application should neither be too slow nor too fast. Too slow loading yields to poor sequencing array coverage, while too fast loading might flush out RNA from the array into the waste sink.
?. [Nanopore Direct RNA library preparation (SQK-RNA002)]
Sequence under the settings recommended for your flow cell (depending on prior use, storage, and kit components; external Link: Start Sequencing)
?. [RNA extraction and quality control]
Add  chloroform per TRIZOL and shake by tube inversion
500 µl
1 ml
?. [RNA extraction and quality control]
Centrifuge  at 12-15,000g at  to separate phases
4 °C
?. [RNA extraction and quality control]
Carefully transfer the aqueous phase to a new Eppendorf Safe-lock 1.5ml tube by angling the tube for most efficient transfer