Available via license: CC BY 4.0
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NOV 17, 2019
ONT DirectRNA Library preparation for poly(A)
estimation
Maximilian Krause ,Adnan M Niazi
University of Bergen
The Nordic Nanopore-Seq Netw ork
Maximilian Krause
University of Bergen
DOI:
dx.doi.org/10.17504/protocol
s.io.9cjh2un
External link:
http://doi:10.1261/rna.07133
2.119
Protocol Citation: Maximilian
Krause, Adnan M Niazi 2019.
ONT DirectRNA Library
preparation for poly(A)
estimation. protocols.io
https://dx.doi.org/10.17504/p
rotocols.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.
1 1
1
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
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Created: Nov 15, 2019
Last Modified: Nov 17, 2019
PROTOCOL integer ID:
29803
Keywords: Nanopore, Direct
RNA Sequencing, poly(A),
tailfindr
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 stoppin g poi nt. NO safe stoppi ng 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 rxns N e w England BiolabsNew England Biol a bs CatalogCatalog
##E6056SE6056S
Qubit dsDNA HS Assay Kit T herm o Fisher Scientifi cTh erm o Fish er Scienti fic C atalog #Catalog # Q32851Q32851
Agencourt RNAClean XP Beads Beckman C oul terBeckman Coulte r Catal og #Ca talog # A63987A63987
PCR Machine Contri bute d by usersContributed by users
95% EtOH C ontribute d by use rsCon tri but ed by users
Nuclease-free water Th erm o Fish er Scienti ficThe rmo Fi she r Sci enti fic Ca talog #Cata log # R0581R0581
Centrifuge 5424 R refrigerated with Rotor FA-45-24-11 rotary knobs 120 V/50 – 60
Hz (US) Eppendorf Centri fugeEppe ndorf C e ntrifuge Cata log #C atal og #54040005375404000537
Qubit Fluorometer Life Te chnol ogi e sLife Technologies Cata log #Catal og #Q33216Q33216
2-Propanol Si gma AldrichSi gma Aldrich C atal og #Ca talog # 190764190764
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DNA LoBind Tubes, 1.5 mL EppendorfEppe ndorf Ca talog #Cata log # 00301080510030108051
Qubit RNA HS Assay Kit T herm o Fisher Scientifi cTh erm o Fish er Scienti fic C atalog #Catalog # Q32852Q32852
Qubit assay tubes The rmo Fi she r Sci e nti ficThermo Fisher S cie ntific Catalog #Catal og #Q32856Q32856
Chloroform Sigma Al dri chSi gma Aldri ch Cata l og #Catalog #372978372978
TRIzol™ LS Reagent The rmo F i she rTh erm o Fish er Cata l og #Catalog #1029602810296028
DynaMag™-2 Magnet Th ermo Fish erTherm o Fisher Cata log #Catal og #12321D12321D
HulaMixer™ Sample Mixer Thermo Fi sherThe rmo Fi she r C atal og #Ca talog # 15920D15920D
SuperScript™ III Reverse Transcriptase The rmo Fi she rThe rmo F ishe r Cata l og #C a talog #1808004418080044
Thin-walled, frosted lid, RNase-free PCR tubes (0.2 mL) T herm o FisherThermo Fisher Ca talogCa talog
## AM12225AM 12225
Poly(A)Purist™ MAG Kit Th ermo Fish erTherm o Fisher Catalog #Catal og #AM 1922AM 1922
GlycoBlue™ Coprecipitant (15 mg/mL) The rmo Fi sherThe rmo Fi she r Catal og #Ca talog # AM9515AM9515
Sodium Acetate (3 M), pH 5.5, RNase-free Therm o FisherTherm o Fisher Cat alog #Cata l og #AM 9740AM 9740
dNTP Mix (10 mM each) The rmo F ishe rTherm o Fisher Cata log #C atalog #R0191R0191
Direct RNA Sequencing kit (SQK-RNA002) Oxford NanoporeOxford Nanopore
Te chn ologiesTe chn ologies C a talog #Cat alog # S QK-RN A002SQK-RNA 002
Flow Cell Priming Kit (EXP-FLP002) Oxford Nanopore Te chnologie sOxford Nanopore Technologies Ca talogCa talog
## EXP -FLP002EXP-FLP 002
MinION sequencer Oxford Nanopore Te chn ologiesOxford Nanopore Technologies
ONT MinION Flow Cell R9.4.1 Oxford N anopore TechnologiesOxford N a nopore Technologies Cata log #C atalog #FLO-FL O-
MIN106DMIN106D
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.
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BEFORE START INSTRUCTIONS
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 safeNO safe
stopping poi nt. stopping poi nt. Thus please make sure you plan with sufficient time for this part of
the experiment
1Resuspend 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
RNA extraction and quality control
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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.
2Incubate 00:05:00 at Room temperature , with regular tube inversion
3Add 200 µL chloroform per 1 mL TRIZOL and shake by tube inversion
4Incubate 00:05:00 at Room temperature , with regular tube inversion
5Centrifuge 00:10:00 at 12-15,000g at 4 °C to separate phases
6Carefully 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.
7Add 500 µL chloroform per 1 mL TRIZOL and shake by tube inversion
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8Centrifuge 00:10:00 at 12-15,000g at 4 °C to separate phases
9Carefully transfer the aqueous phase to a new Eppendorf Safe-lock 1.5ml tube by angling the
tube for most efficient transfer
10 Add 1 µL GlycoBlue reagent, 0.1 Vol 3 Molarity (M) NaOAc and 1 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 and
500 µL
Isopropanol
11 Incubate 01:00:00 at -20 °C for most efficient yields
12 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
13 Aspirate the supernatant without disturbing the RNA pellet
14 Wash the RNA pellet with 1 mL freshly-prepared 75 % volume EtOH
1h
10m
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15 Centrifuge for 00:10:00 at 15-20,000g at 4 °C
16 Aspirate supernatant and repeat ethanol wash go to step #13 once
17 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
18 Add 50 µL of RNase-free water and resuspend by tapping the tube or shaking in a
thermoshaker at Room temperature
19 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.
NanodropNanodrop 260/280 and 260/230 measurements are important to assess possible remnant
contaminants that are detrimental to Nanopore's sequencing chemistry.
Qubi tQubi t measurements offer the most sensitive RNA quantification, and are regularly used
during the library preparation protocols.
BioAnalyze rBioAnalyze r traces yield an RNA integrity measurement that allows to assess the biological
quality of the sample
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20 Bring RNA concentration to 600 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
21 Add an equal volume of the Poly(A)Purist Mag Kit 2x Binding Solution (minimal 50 µL ) and
mix by tube inversion
22 Store RNA On ice until further processing
23 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
24 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
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25 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
26 Repeat once from go to step #24
27 Add the total RNA sample + Binding Solution from go to step #22 to the beads, mix by tube
inversion
28 Heat the bead-RNA mixture to 65 °C for 00:05:00
Note
Longer time and higher temperatures are not advised, to avoid additional RNA degradation
29 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
30 Meanwhile preheat the Kit THE elution buffer to 70 °C
31 Precipitate the magnetic beads with the RNA attached on the magnetic stand and aspirate
supernatant
32 Take the tube out of the magnetic stand and resuspend the beads in Kit Wash Solu tion 1 Wash Sol ution 1 with
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volume depending on go to step #23
33 Repeat once from go to step #31
34 Take the tube out of the magnetic stand and resuspend the beads in Kit Wash Solu tion 2 Wash Sol ution 2 with
volume depending on go to step #23
35 Precipitate beads on the magnetic stand and aspirate the supernatant
36 Repeat o go to step #34
37 Briefly spin down on a tabletop centrifuge to remove residual Wash Solution
38 Remove the tube from the magnetic stand and resuspend in 100 µL hot THE buffer from
go to step #30
39 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
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40 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
41 Repeat from go to step #38 and pool the supernatants into one tube
42 Put the fresh sample tube on a magnet again to collect residual beads for 00:03:00
On ice
43 Transfer the cleaned supernatant into a fresh Eppendorf 1.5ml tube
44 Add 1 µL GlycoBlue reagent, 20 µL 3 Molarity (M) NaAc and 250 µL
Isopropanol, mix by inversion
45 Store 01:00:00 at -20 °C
46 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.
3m
1h
10m
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47 Aspirate the supernatant without disturbing the RNA pellet
48 Wash the RNA pellet with 1 mL freshly-prepared 75 % volume EtOH
49 Centrifuge for 00:10:00 at 15-20,000g at 4 °C
50 Repeat once from go to step #48
51 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
52 Add 15 µL of RNase-free water and resuspend by tapping the tube or shaking in a
thermoshaker at 25 °C .
53 Record quantity and quality by Nanodrop measurement and Qubit RNA Broad Range kit. Test
rRNA removal by BioAnalyzer RNA chip.
10m
2m
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Note
All measurements are necessary for Nanopore Experiments.
NanodropNanodrop 260/280 and 260/230 measurements are important to assess possible remnant
contaminants that are detrimental to Nanopores sequencing chemistry.
Qubi tQubi t measurements offer the most sensitive RNA quantification, and are regularly used
during the library preparation protocols.
BioAnalyze r BioAnalyze r 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.
54 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
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
If your RNA concentration is too low and upconcentration is necessary, use RNAClean XP
bead procedures to increase the concentration of your RNA.
55 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 )
Nanopore Direct RNA library preparation (SQK-RNA002)
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56 (optionally) add 0.5 µL RNA CS from the Nanopore kit to monitor sequencing quality
57 Incubate for 00:15:00 at Room temperature
Note
Longer time can increase ligation efficiency, yet increase the chance of further RNA
degradation
58 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
59 After RNA incubation, add the Master Mix to the RNA sample and mix by careful pipetting
60 Add 2 µL SuperScript III RT enzyme and mix by careful pipetting
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61 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
62 Transfer whole volume into a fresh Eppendorff 1.5ml Lo-Bind safe-lock tube
Note
It is extremel y im portan textre m e ly i mporta nt 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!
63 Add 72 µL RNAClean XP beads and resuspend by careful pipetting
64 Incubate at Room temperature under constant agitation for 00:10:00
Note
Every in cubati on stepEvery in cubati on 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.
65 Pellet beads on a magnetic stand and aspirate supernatant
1h
10m
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66 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
67 Aspirate EtOH, spin down briefly on tabletop centrifuge and remove residual EtOH
68 Resuspend beads with 20 µL RNase-free water by tapping the tube
69 Incubate 00:10:00 at Room temperature
70 Pellet beads on the magnetic stand and transfer to a new 1.5ml Lo-bind Safe-lock tube
71 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 )
72 Carefully mix by pipetting and incubate 00:15:00 at Room temperature
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73 Add 40 µL RNAClean XP beads and resuspend by careful pipetting
74 Incubate at Room temperature under constant agitation for 00:10:00
75 Pellet beads on a magnetic stand and aspirate supernatant
76 Wash the beads on the magnet with 150 µL Nanopore Wash Buffer (WSB) by resuspending
the beads by tube-flicking
77 Aspirate Wash Buffer and repeat washing go to step #76
78 Aspirate Wash Buffer, spin down briefly on tabletop centrifuge and remove residual liquid
79 Resuspend beads with 21 µL Elution buffer water by tapping the tube
80 Incubate 00:10:00 at Room temperature
81 Pellet beads on the magnetic stand and transfer to a new 1.5ml Lo-bind Safe-lock tube
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82 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
83 Add 17.5 µL RNase-free water and 37.5 µL Nanopore Sequencing Buffer (RRB) to the
library
84 Prime a MinION flow cell as specified in Nanopore protocols, and finally load the library drop-wise
through the Sample portSampl e port (a detailed description including video documentation can be found
here: Flow Cell Priming)
Note
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.
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.
85 Sequence under the settings recommended for your flow cell (depending on prior use, storage,
and kit components; external Link: Start Sequencing)
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