Nov 17, 2019
ONT DirectRNA Library preparation for poly(A) estimation
- 1University of Bergen
External link: http://doi:10.1261/rna.071332.119
Protocol Citation: Maximilian Krause, Adnan M Niazi 2019. ONT DirectRNA Library preparation for poly(A) estimation. protocols.io https://dx.doi.org/10.17504/protocols.io.9cjh2un
Manuscript citation:
Krause M, Niazi AM, Labun K, Torres Cleuren YN, Müller FS, Valen E.tailfindr: alignment-free poly(A) length measurement for Oxford Nanopore RNA and DNA sequencing.RNA. 2019;25(10):1229–1241. doi:10.1261/rna.071332.119
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
We use this protocol in our group and it is working.
Created: November 15, 2019
Last Modified: November 17, 2019
Protocol Integer ID: 29803
Keywords: Nanopore, Direct RNA Sequencing, poly(A), tailfindr
Abstract
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.
The protocol is used to assess poly(A) tail length using the tailfindr 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 tailfindr visit the publication or the GitHub repository
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)Eppendorf CentrifugeCatalog #5404000537
Qubit FluorometerLife TechnologiesCatalog #Q33216
2-PropanolSigma AldrichCatalog #190764
DNA LoBind Tubes, 1.5 mLEppendorfCatalog #0030108051
Qubit RNA HS Assay KitThermo Fisher ScientificCatalog #Q32852
Qubit assay tubesThermo Fisher ScientificCatalog #Q32856
ChloroformSigma AldrichCatalog #372978
TRIzol™ LS ReagentThermo FisherCatalog #10296028
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
Poly(A)Purist™ MAG KitThermo FisherCatalog #AM1922
GlycoBlue™ Coprecipitant (15 mg/mL)Thermo FisherCatalog #AM9515
Sodium Acetate (3 M), pH 5.5, RNase-freeThermo FisherCatalog #AM9740
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 MinION Flow Cell R9.4.1Oxford Nanopore TechnologiesCatalog #FLO-MIN106D
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. 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 500ng. As mRNA is routinely only 1% of total RNA, it should be aimed for extracting 25ug of total RNA from the sample.
Extraction should be chosen to avoid any contaminants, as these could be detrimental to the sequencing chemistry. In our experience, silica-column based purification strategies not only cause RNA degradation by physical force, but also are prone to retain Guanidine-hydrochloride contamination. We thus advise on the use of phenol-chloroform extraction methods, such as the use of TRI reagent. These are more time-consuming, but in our hands yield higher quality RNA with minimal contaminant carry-over.
Poly(A) enrichment (or any small RNA depletion strategy) is necessary to ensure efficient sequencing analysis, as the essential Motor Protein is added to the RNA via poly(A)-guided ligation. Non-poly(A)-containing RNA thus acts as an inert contaminant that affects proper sequencing. We routinely use the Poly(A)Purist MAG Kit, but any other strategies that do not involve vortexing, vigorous pipetting or column-based purification would work as well.
Described below is the full workflow from total RNA to sequencing using TRI reagent and the Poly(A)Purist MAG kit.
After poly(A) RNA enrichment, the Library preparation protocol has NO safe stopping point. Thus please make sure you plan with sufficient time for this part of the experiment
RNA extraction and quality control
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
Note
Homogenization should be kept as gentle as possible to avoid RNA molecule degradation. Reduce number of pestle strokes, pipetting, or replace by vigorous shaking.
Incubate 00:05:00 at Room temperature , with regular tube inversion
5m
Add 200 µL chloroform per 1 mL TRIZOL and shake by tube inversion
Incubate 00:05:00 at Room temperature , with regular tube inversion
5m
Centrifuge 00:10:00 at 12-15,000g at 4 °C to separate phases
10m
Carefully transfer the aqueous phase to a new Eppendorf Safe-lock 1.5ml tube by angling the tube for most efficient transfer
Note
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.
Add 500 µL chloroform per 1 mL TRIZOL and shake by tube inversion
Centrifuge 00:10:00 at 12-15,000g at 4 °C to separate phases
Carefully transfer the aqueous phase to a new Eppendorf Safe-lock 1.5ml tube by angling the tube for most efficient transfer
Add 1 µL GlycoBlue reagent, 0.1 Vol 3 Molarity (M) NaOAc and1 Vol Isopropanol and mix by inversion of the tube
By experience, samples with an initial volume of 1ml TRI reagent will need 50 µL 3 Molarity (M) NaOAc and500 µL Isopropanol
Incubate 01:00:00 at -20 °C for most efficient yields
1h
Centrifuge for 00:10:00 at 15-20,000g at 4 °C
Note
Total RNA should form a strong white pellet. Care should be taken to not aspirate the pellet during the following washing steps
10m
Aspirate the supernatant without disturbing the RNA pellet
Wash the RNA pellet with 1 mL freshly-prepared 75 % volume EtOH
Centrifuge for 00:10:00 at 15-20,000g at 4 °C
10m
Aspirate supernatant and repeat ethanol wash go to step #13 once
Aspirate the supernatant and air-dry the pellet for 00:05:00 can be reduced to 2 min
Note
If necessary, briefly spin down on a tabletop centrifuge to collect remaining EtOH, and pipet off with a 200ul pipet
5m
Add 50 µL of RNase-free water and resuspend by tapping the tube or shaking in a thermoshaker at Room temperature
Record quantity and quality by Nanodrop measurement and Qubit RNA Broad Range kit. Test RNA integrity by BioAnalyzer RNA chip
Note
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
poly(A) enrichment
Bring RNA concentration to600 ng/ul , but minimal 50 µL (30 µg RNA)
Note
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
Add an equal volume of the Poly(A)Purist Mag Kit 2x Binding Solution (minimal50 µL ) and mix by tube inversion
Store RNA On ice until further processing
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 100 µg of total RNA from above, use 10 µL Magnetic Beads solution and in subsequent washing steps 50 µL Wash Buffer
Note
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
Precipitate the beads on a magnetic stand and aspirate the buffer
Note
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
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, go to step #23
Repeat once from go to step #24
Add the total RNA sample + Binding Solution from go to step #22 to the beads, mix by tube inversion
Heat the bead-RNA mixture to65 °C for 00:05:00
Note
Longer time and higher temperatures are not advised, to avoid additional RNA degradation
5m
Incubate 00:30:00 at Room temperature under constant agitation
Note
Longer incubation time to up to 1h is possible, but increases the chance of RNA degradation
30m
Meanwhile preheat the Kit THE elution buffer to 70 °C
Precipitate the magnetic beads with the RNA attached on the magnetic stand and aspirate supernatant
Take the tube out of the magnetic stand and resuspend the beads in Kit Wash Solution 1 with volume depending on go to step #23
Repeat once from go to step #31
Take the tube out of the magnetic stand and resuspend the beads in Kit Wash Solution 2 with volume depending on go to step #23
Precipitate beads on the magnetic stand and aspirate the supernatant
Repeat ogo to step #34
Briefly spin down on a tabletop centrifuge to remove residual Wash Solution
Remove the tube from the magnetic stand and resuspend in 100 µL hot THE buffer from go to step #30
Incubate 00:01:00 can be extended to 2 min at 70 °C
Note
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.
2m
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
Repeat from go to step #38 and pool the supernatants into one tube
Put the fresh sample tube on a magnet again to collect residual beads for 00:03:00 On ice
3m
Transfer the cleaned supernatant into a fresh Eppendorf 1.5ml tube
Add 1 µL GlycoBlue reagent, 20 µL 3 Molarity (M) NaAc and 250 µL Isopropanol, mix by inversion
Store 01:00:00 at -20 °C
1h
Centrifuge for 00:10:00 at 15-20,000g at 4 °C
Note
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.
10m
Aspirate the supernatant without disturbing the RNA pellet
Wash the RNA pellet with 1 mL freshly-prepared 75 % volume EtOH
Centrifuge for 00:10:00 at 15-20,000g at 4 °C
10m
Repeat once from go to step #48
Aspirate the supernatant and air-dry the pellet for 00:02:00
Note
If necessary, briefly spin down on a tabletop centrifuge to collect remaining EtOH, and pipet off with a 200ul pipet
2m
Add 15 µL of RNase-free water and resuspend by tapping the tube or shaking in a thermoshaker at 25 °C .
Record quantity and quality by Nanodrop measurement and Qubit RNA Broad Range kit. Test rRNA removal by BioAnalyzer RNA chip.
Note
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)
Nanopore Direct RNA library preparation (SQK-RNA002)
Take 500 ng poly(A)-selected RNA into a 0.2ml thin-walled DNA-free PCR tube and bring volume to 9 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:
- 1 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 20 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:
- 8 µL NEBNext Quick Ligation buffer
- 6 µL Nanopore RNA Adapter Mix (RMX)
- 3 µL RNase-free water
- 3 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 40 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 150 µL Nanopore Wash Buffer (WSB) by resuspending the beads by tube-flicking
Aspirate Wash Buffer and repeat washing go to step #76
Aspirate Wash Buffer, spin down briefly on tabletop centrifuge and remove residual liquid
Resuspend beads with 21 µL Elution buffer 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
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
Add 17.5 µL RNase-free water and 37.5 µL Nanopore Sequencing Buffer (RRB) to the library
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)
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
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.
Sequence under the settings recommended for your flow cell (depending on prior use, storage, and kit components; external Link: Start Sequencing)