Feb 10, 2023

Public workspaceRCA-NGS for RNA viruses

DOI
dx.doi.org/10.17504/protocols.io.rm7vzb7wrvx1/v1
  • Masayasu Misu1,2,
  • Tomoki Yoshikawa1,
  • Satoko Sugimoto1,
  • Yuki Takamatsu1,
  • Takeshi Kurosu1,
  • Yukiteru Ouji2,
  • Masahide Yoshikawa2,
  • Masayuki Shimojima1,
  • Hideki Ebihara1,
  • Masayuki Saijo1
  • 1Department of Virology I, National Institute of Infectious Diseases;
  • 2Department of Pathogen, Infection and Immunity, Nara Medical University
Masayasu Misu
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DOI: dx.doi.org/10.17504/protocols.io.rm7vzb7wrvx1/v1
Protocol Citation: Masayasu Misu, Tomoki Yoshikawa, Satoko Sugimoto, Yuki Takamatsu, Takeshi Kurosu, Yukiteru Ouji, Masahide Yoshikawa, Masayuki Shimojima, Hideki Ebihara, Masayuki Saijo 2023. RCA-NGS for RNA viruses. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzb7wrvx1/v1
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 and it's working
Created: January 29, 2023
Last Modified: February 10, 2023
Protocol Integer ID: 76026
Keywords: Oxford Nanopore Technology, RNA virus, Sequence method, MinION, Nanopore sequencing, RCA-NGS
Abstract
This RCA-NGS were optimized for an NGS machine, MinION. These methods do not require nucleic acid amplification with virus-specific PCR primers, physical viral particle enrichment, and RACE.

These methods enable whole RNA viral genome sequencing by combining the following techniques:
1) removal of unwanted DNA and RNA other than the RNA viral genome by nuclease treatment
2) the terminal of viral genome sequence determination by barcoded linkers ligation
3) Amplification of the viral genomic cDNA using an isothermal DNA amplification technique, such as rolling circle amplification (RCA).

This method can be exploited to determine any whole RNA viral genomes (i.e., single-stranded, double-stranded, positive-stranded, negative-stranded, non-segmented or multi-segmented genomes).
Materials
  • ReagentMicrococcal Nuclease - 320,000 gel unitsNew England BiolabsCatalog #M0247S
  • ReagentHigh Pure Viral RNA KitRocheCatalog #11858882001
  • ReagentTurbo DNA-free KitInvitrogen - Thermo FisherCatalog #AM1907
  • NucleoSpin RNA Clean-up XS - Takara, Catalog #740903.10
  • ReagentT4 RNA Ligase 2, truncated KQ - 2,000 unitsNew England BiolabsCatalog #M0373S
  • The barcode-polyA linker DNA (e.g., The cSP6-polyA linker DNA)
  • ReagentSuperscript IVThermo Fisher ScientificCatalog #18090050
  • SP6 primer (e.g., 5′ phosphorylated SP6 primer)
  • ReagentDeoxynucleotide (dNTP) Solution MixNew England BiolabsCatalog #N0447S
  • ReagentSuperase-In RNase InhibitorThermofisherCatalog #AM2694
  • Dr.GenTLE Precipitation Carrier - Takara Catalog #9094
  • ReagentRNase H - 250 unitsNew England BiolabsCatalog #M0297S
  • ReagentAgencourt AMPure XPBeckman CoulterCatalog #A63880
  • CircLigase II ssDNA Ligase - Biosearch Technologies Catalog #CL9021K
  • GenomiPhi V3 Ready-To-Go DNA Amplification Kit - Cytiva Catalog #25-6601-24
  • ReagentT7 Endonuclease I - 250 unitsNew England BiolabsCatalog #M0302S
  • ReagentNEBNext FFPE DNA Repair Mix - 24 rxnsNew England BiolabsCatalog #M6630S
  • ReagentNEBNext Ultra II End Repair/dA-Tailing Module - 24 rxnsNew England BiolabsCatalog #E7546S
  • ReagentBlunt/TA Ligase Master Mix - 50 rxnsNew England BiolabsCatalog #M0367S
  • ReagentNEBNext Quick Ligation Module - 20 rxnsNew England BiolabsCatalog #E6056S
  • Ligation Sequencing Kit - Oxford Nanopore Technologies Catalog #SQK-LSK109
  • Native Barcoding Expansion - Oxford Nanopore Technologies Catalog #EXP-NBD104, #EXP-NBD114
  • ReagentQubit 4 FluorometerThermo Fisher ScientificCatalog #Q33238
  • ReagentQubit 1X dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q33230
  • ReagentDNA LoBind Tube 1.5ml EppendorfCatalog #022431021
  • Reagent0.2 ml PCR Tube stripsEppendorfCatalog #0030124359
  • 100 % ethanol
  • 70 % ethanol
  • TE(pH8.0)
  • nuclease-free H2O




Protocol materials
ReagentMicrococcal Nuclease - 320,000 gel unitsNew England BiolabsCatalog #M0247S
Materials, Step 3
ReagentNEBNext Quick Ligation Module - 20 rxnsNew England BiolabsCatalog #E6056S
Materials, Step 29
ReagentHigh Pure Viral RNA KitRocheCatalog #11858882001
Materials, Step 4
ReagentQubit 1X dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q33230
In Materials and 4 steps
ReagentT7 Endonuclease I - 250 unitsNew England BiolabsCatalog #M0302S
Materials, Step 19
ReagentSuperscript IVThermo Fisher ScientificCatalog #18090050
Materials, Step 9
ReagentNEBNext Ultra II End Repair/dA-Tailing Module - 24 rxnsNew England BiolabsCatalog #E7546S
Materials, Step 21
ReagentNEBNext FFPE DNA Repair Mix - 24 rxnsNew England BiolabsCatalog #M6630S
Materials, Step 21
Reagent0.2 ml PCR Tube stripsEppendorfCatalog #0030124359
Materials, Step 6.5
ReagentRNase H - 250 unitsNew England BiolabsCatalog #M0297S
Materials, Step 10
ReagentBlunt/TA Ligase Master Mix - 50 rxnsNew England BiolabsCatalog #M0367S
Materials, Step 24
ReagentDeoxynucleotide (dNTP) Solution MixNew England BiolabsCatalog #N0447S
Materials, Step 9.1
ReagentT4 RNA Ligase 2, truncated KQ - 2,000 unitsNew England BiolabsCatalog #M0373S
Materials, Step 7
ReagentSuperase-In RNase InhibitorThermofisherCatalog #AM2694
Materials, Step 9.2
ReagentDNA LoBind Tube 1.5ml EppendorfCatalog #022431021
Materials, Step 4.5
ReagentQubit 4 FluorometerThermo Fisher ScientificCatalog #Q33238
Materials
ReagentAgencourt AMPure XPBeckman CoulterCatalog #A63880
In Materials and 7 steps
ReagentTurbo DNA-free KitInvitrogen - Thermo FisherCatalog #AM1907
Materials, Step 5
Safety warnings
Follow your facility's regulations and biosafety practices.
Before start
This method was only confirmed to work with the working stocks that contain isolated RNA viruses at least 3.0 × 105 TCID50 per ml.
It is recommended to check no bacterial contamination(e.g., Mycoplasma spp.).
Preparation for virus supernatant
Preparation for virus supernatant
Centrifuge the working stock virus to remove debris.
Centrifigation6000 x g, Room temperature, 00:10:00

10m
Transfer Amount180 µL virus supernatant to a 1.5ml screw cap tube.
Unwanted DNA and RNA mainly originating from the virus-infected cells are digested usingReagentMicrococcal Nuclease - 320,000 gel unitsNew England BiolabsCatalog #M0247S .
Total 201 μl reaction

  • Amount180 µL virus supernatant
  • Amount20 µL 10X Micrococcal Nuclease Reaction Buffer
  • Amount1 µL Micrococcal nuclease

Mix by pipetting and spin down.
Temperature37 °C water bath Duration01:00:00


1h
The viral genomic RNA extraction
The viral genomic RNA extraction
The viral genomic RNA extraction is performed using ReagentHigh Pure Viral RNA KitRocheCatalog #11858882001 .
Add Amount400 µL of binding buffer (with Amount4 µL PolyA carrier RNA).

Mix gently by ~5 times pipetting and flicking thoroughly the tube, and spin down.
TemperatureRoom temperature Duration00:10:00
10m
Transfer the sample to a High Pure Filter Tube.
Centrifigation8000 x g, Room temperature, 00:01:00

Discard the flow-through liquid and Collection Tube, and insert the Filter Tube into a new Collection Tube.
1m
Add Amount500 µL of inhibitor removal bo transfer the sample to a High Pure Filter Tube.
Centrifigation8000 x g, Room temperature, 00:01:00

Discard the flow-through liquid and Collection Tube, and insert the Filter Tube into a new Collection Tube.
1m
Add Amount450 µL of wash buffer.
Centrifigation8000 x g, Room temperature, 00:01:00

Discard the flow-through liquid and Collection Tube, and insert the Filter Tube into a new Collection Tube.
1m
Add Amount450 µL of wash buffer.
Centrifigation13000 x g, Room temperature, 00:01:00 and discard the flow-through liquid.

Discard the Collection Tube and insert the Filter Tube into a 1.5 ml tube -ReagentDNA LoBind Tube 1.5ml EppendorfCatalog #022431021 .

1m
Add Amount50 µL Elution Buffer.
Centrifigation13000 x g, Room temperature, 00:01:00

Note
The eluted RNA can be stored at -80℃.

1m
Remove unwanted DNA
Remove unwanted DNA
Unwanted DNA mainly from the virus-infected cells in the RNA sample is digested using a ReagentTurbo DNA-free KitInvitrogen - Thermo FisherCatalog #AM1907 .
Total 56 μl reaction

  • Amount50 µL the eluted RNA
  • Amount5 µL 10X reaction buffer
  • Amount1 µL DNase I

Mix gently by pipetting and spin down.
Temperature37 °C Duration00:30:00
30m
The viral RNA is purified using NucleoSpin RNA Clean-up XS - Takara, Catalog #740903.10.
Add an equal volume Amount56 µL of Buffer RCU and mix gently.

Transfer the sample to a NucleoSpin RNA XS Column.
Centrifigation11000 x g, Room temperature, 00:01:00
1m
Wash the column by Amount400 µL Buffer RA3.
Centrifigation11000 x g, Room temperature, 00:01:00

Discard the flow-through liquid and Collection Tube, and insert the NucleoSpin RNA XS Column into a new Collection Tube.
1m
Wash the column by Amount200 µL Buffer RA3.
Centrifigation11000 x g, Room temperature, 00:02:00

Discard the flow-through liquid and Collection Tube, and insert the NucleoSpin RNA XS Column into a Nuclease-free Collection Tube(1.5 ml).
2m
Add Amount10 µL RNase-free H2O.
Centrifigation11000 x g, Room temperature, 00:01:00
Transfer the sample to a 0.2 ml PCR tube -Reagent0.2 ml PCR Tube stripsEppendorfCatalog #0030124359 .

1m
cSP6-polyA Linker DNA ligation
cSP6-polyA Linker DNA ligation

The viral RNA is ligated with cSP6-polyA Linker DNA usingReagentT4 RNA Ligase 2, truncated KQ - 2,000 unitsNew England BiolabsCatalog #M0373S .
The RNA is ligated to the 3' end with the barcoded(complementary sequence of SP6 (cSP6)) polyA linker DNA. It is able to identify the 3’ terminal viral genome sequence. The PolyA sequence is required for reverse transcription for ONT kit (SQK-PBK004/ PCS109).
Note
The cSP6-polyA linker DNA (5'-5rApp-CTATAGTGTCACCTAAATCAAAAAAAAAAAAAAAAAAAA-3ddC-3'), which is pre-adenylated at the 5' terminal (5rApp), and consists of the complementary sequence of SP6 (CTATAGTGTCACCTAAATC), oligo (dA) 20, and dideoxycytidine (3ddC) at the 3' terminal, was synthesised for 3' linker ligation by Integrated DNA Technologies (Coralville, IA).




Total 20 μl reaction

  • Amount10 µL Purified RNA
  • Amount1 µL 10 μM the cSP6-polyA linker DNA
  • Amount2 µL 10X T4 RNA Ligase Reaction Buffer
  • Amount6 µL 50% PEG8000 solution
  • Amount1 µL T4 RNA Ligase 2, truncated KQ

Mix gently by pipetting and spin down.
Incubate Temperature25 °C Duration00:15:00

15m
The linker-ligated viral RNA is purified using NucleoSpin RNA Clean-up XS - Takara, Catalog #740903.10
Go togo to step #6

Fill the sample to 100 μl with 80 μl TE (pH 8.0) and add 100 μl (equal volume) of Buffer RCU.

Eluted the RNA in Amount10 µL RNase-free H2O and transfer the sample to a 0.2 ml PCR tube.

Reverse transcription
Reverse transcription
The viral RNA is reverse transcribed using ReagentSuperscript IVThermo Fisher ScientificCatalog #18090050 .
5′ phosphorylated SP6 primer is used for reverse transcription.
Note
SP6 primer (5′ phosphorylated SP6 primer); 5' [Phos]GATTTAGGTGACACTATAG 3'
5' phosphorylation is due to circularization.


Set up pre-mixture

  • Amount10 µL RNA (~ 50ng)
  • Amount1 µL 50 μM SP6 primer
  • Amount1 µL nuclease-free H2O
  • Amount1 µL 10mM dNTP - ReagentDeoxynucleotide (dNTP) Solution MixNew England BiolabsCatalog #N0447S

Mix gently by flicking the tube, and spin down.
Temperature65 °C Duration00:05:00 and Temperature4 °C Duration00:01:00




6m
Total 20 μl reaction

  • Amount13 µL pre-mixture sample
  • Amount4 µL 5X SSIV Buffer
  • Amount1 µL 100mM DTT
  • Amount1 µL RNase OUT - ReagentSuperase-In RNase InhibitorThermofisherCatalog #AM2694
  • Amount1 µL SuperScript IV Reverse Transcriptase

Mix gently by flicking the tube, and spin down.
Temperature55 °C Duration00:10:00
Temperature80 °C Duration00:10:00


20m
RNase H treatment and ethanol precipitation
RNase H treatment and ethanol precipitation
20m
20m
Add Amount1 µL ReagentRNase H - 250 unitsNew England BiolabsCatalog #M0297S . Temperature37 °C Duration00:20:00

20m
Ethanol precipitation using Dr.GenTLE Precipitation Carrier - Takara Bio Catalog #9094.
Add Amount20 µL TE(pH8.0) to fill up the sample to Amount40 µL .
.
Add Amount4 µL 3M CH3COONa (pH5.2) .

Add Amount1 µL Dr.GenTLE Precipitation Carrier.

Add Amount100 µL 100% ethanol.
Centrifigation13000 x g, Room temperature, 00:15:00

15m
Discard the supernatant.
Wash the pellet with Amount500 µL 70% ethanol.
Centrifigation13000 x g, Room temperature, 00:05:00
5m
Discard the supernatant and dry.
Dissolve the pellet in Amount12 µL nuclease-free H2O.
(Optional step) Short cDNA fragment removal instead of Ethanol precipitation (#11)
(Optional step) Short cDNA fragment removal instead of Ethanol precipitation (#11)
Short cDNA fragment is removed from the viral RNA sample using ReagentAgencourt AMPure XPBeckman CoulterCatalog #A63880
Prepare AMpure XP beads for use; resuspend by vortexing.
Transfer amplified DNA sample to 1.5ml low binding tube.

Note
If a significant proportion of the reads obtained from an NGS run fail to match with the NCBI-nr database (i.e., no hits), it could indicate a large number of short cDNA fragments in the sample. In such instances, re-performing the optional step instead of step 11, such as ethanol precipitation could significantly enhance the outcomes.

Add Amount36 µL (X1.8 volume) AMPure XP reagent and mix by pipetting.
Incubate on rotor mixer.
Duration00:05:00 TemperatureRoom temperature
Spin down and pellet on a magnet.
Wait for Duration00:01:00 and pipette off the supernatant.
Wash twice by Amount200 µL 70 % ethanol and remove the ethanol using a pipette and discard.
Spin down and pipette off any residual ethanol.
Resuspend pellet in Amount20 µL TE(pH 8.0).
Temperature37 °C Duration00:03:00 and tapping occasionally.
Incubate on a rotor mixer.
Duration00:07:00
Spin down and pellet the beads on the magnet until the elute is clear and colourless.
Remove retain Amount20 µL elute into a new tube.
Size selection of the cDNA sample is performed using ReagentAgencourt AMPure XPBeckman CoulterCatalog #A63880 .
X0.8 volume of AMPure beads recovers more than 200 bp of nucleic acids.
Add Amount16 µL (X0.8 volume) AMPure beads and mix by pipetting.
Incubate on rotor mixer.
Duration00:05:00 TemperatureRoom temperature
Spin down and pellet on a magnet.
Wait for Duration00:01:00 and pipette off the supernatant.
Wash twice by Amount200 µL 70 % ethanol and remove the ethanol using a pipette and discard.
Spin down and pipette off any residual ethanol.
Resuspend pellet in Amount12 µL nuclease-free water.
Temperature37 °C Duration00:03:00 and tapping occasionally.
Incubate on a rotor mixer.
Duration00:07:00
Spin down and pellet the beads on the magnet until the elute is clear and colourless.
Remove retain Amount12 µL elute into a new tube.
Circularization of cDNA
Circularization of cDNA
1h 10m
1h 10m
The cDNA is circularized using CircLigase II ssDNA Ligase - Biosearch Technologies Catalog #CL9021K.
Total 20 μl reaction

  • Amount12 µL cDNA
  • Amount2 µL 10X reaction buffer
  • Amount1 µL 50 mM MnCl2
  • Amount4 µL 5M Betaine
  • Amount1 µL CircLigase II
Mix by pipetting and spin down.
Temperature60 °C Duration01:00:00
Temperature80 °C Duration00:10:00

1h 10m
Ethanol precipitation using Dr.GenTLE Precipitation Carrier - Takara Catalog #9094.
Go togo to step #11
Dissolve the pellet in Amount10 µL nuclease-free H2O.
Amplification of cDNA by rolling circle amplification (RCA)
Amplification of cDNA by rolling circle amplification (RCA)
cDNA is amplified by Rolling circle amplification (RCA) using GenomiPhi V3 Ready-To-Go DNA Amplification Kit - Cytiva Catalog #25-6601-24.

Total 20 μl reaction

  • Amount10 µL cDNA
  • Amount10 µL 2X denaturation buffer

Mix by pipetting and spin down.
Temperature95 °C Duration00:03:00
Temperature4 °C on ice
3m
Add 20 μl denatured sample to Ready to go GenomiPhi cake.

Temperature30 °C Duration04:00:00
Temperature65 °C Duration00:10:00

4h 10m
The cDNA is purified by ReagentAgencourt AMPure XPBeckman CoulterCatalog #A63880
Prepare AMpure XP beads for use; resuspend by vortexing.
Transfer amplified DNA sample to 1.5ml low binding tube.

Add Amount36 µL (X1.8 volume) AMPure beads and mix by pipetting.
Incubate on rotor mixer.
Duration00:05:00 TemperatureRoom temperature
Spin down and pellet on a magnet and wait for Duration00:01:00 and pipette off the supernatant.
Wash twice by Amount200 µL 70 % ethanol and remove the ethanol using a pipette and discard.
Spin down and pipette off any residual ethanol.
Resuspend pellet in Amount40 µL nuclease-free H2O.
Temperature37 °C Duration00:03:00 and tapping occasionally.
Incubate on a rotor mixer.
Duration00:07:00
Spin down and pellet the beads on the magnet until the elute is clear and colourless.
Remove retain Amount40 µL elute into a new tube.
DNA concentration is measured using a Qubit 4 Fluorometer with ReagentQubit 1X dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q33230 .

  • Amount199 µL 1X working solution
  • Amount1 µL DNA

Mix by vortexing.

Incubate Duration00:02:00 TemperatureRoom temperature and measure.

Note
Confirm the total amplified cDNA to be over 1500 ng, as confirmed using, for instance, a Qubit 4 Fluorometer and Qubit 1X dsDNA HS Assay Kit.

T7 endonuclease treatment
T7 endonuclease treatment
The amplified cDNA by RCA is digested using ReagentT7 Endonuclease I - 250 unitsNew England BiolabsCatalog #M0302S to remove branching.

The following protocol is modified based on the Native barcoding amplicons (with EXP-NBD104, EXPNBD114,and SQK-LSK109) protocol (NBA_9093_v109_revA_12Nov2019) provided by Oxford Nanopore Technologies website.
Total 30 μl reaction

  • Amountx µL (1.0 μg) DNA
  • Amount3 µL NEBuffer 2
  • Amount1.5 µL T7 endonuclease I
  • Amount25-x µL nuclease-free H2O

Mix by pipetting and spin down.
Temperature37 °C Duration00:30:00
30m
The cDNA is purified using ReagentAgencourt AMPure XPBeckman CoulterCatalog #A63880 .
Go to (Add Amount54 µL (X1.8 volume) AMPure beads)

Resuspend pellet in Amount24 µL nuclease-free H2O.
DNA repair and end-prep
DNA repair and end-prep
The purified cDNA is end-prepped using
ReagentNEBNext FFPE DNA Repair Mix - 24 rxnsNew England BiolabsCatalog #M6630S and
ReagentNEBNext Ultra II End Repair/dA-Tailing Module - 24 rxnsNew England BiolabsCatalog #E7546S
Total 30 μl reaction

  • Amount24 µL DNA
  • Amount1.75 µL NEB Next FFPE DNA repair buffer
  • Amount1 µL NEB Next FFPE DNA repair Mix
  • Amount1.75 µL Ultra II end-prep reaction buffer
  • Amount1.5 µL Ultra II end-prep reaction Mix

Mix by pipetting and spin down.
Temperature20 °C Duration00:30:00
Temperature65 °C Duration00:05:00
35m
The cDNA is purified using ReagentAgencourt AMPure XPBeckman CoulterCatalog #A63880 .
Go to (Add Amount54 µL (X1.8 volume) AMPure beads)

Resuspend pellet in Amount30 µL nuclease-free H2O.
DNA concentration is measured using a Qubit 4 Fluorometer with ReagentQubit 1X dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q33230 .

  • Amount199 µL 1X working solution
  • Amount1 µL DNA

Mix by vortexing.

Incubate Duration00:02:00 TemperatureRoom temperature and measure.

Note
Confirm the purified cDNA to be approximately 700 ng or more using, for instance, Qubit 4 Fluorometer with a Qubit 1X dsDNA HS Assay Kit.

2m
Native barcode ligation
Native barcode ligation
The end-prepped cDNA is ligated with native barcode using Native Barcoding Expansion - Oxford Nanopore Technologies Catalog #EXP-NBD104, #EXP-NBD114 and ReagentBlunt/TA Ligase Master Mix - 50 rxnsNew England BiolabsCatalog #M0367S .
Total 25 μl reaction

  • Amountx µL DNA(about 400ng)
  • Amount1.5 µL native barcode
  • Amount12.5 µL Blunt/TA ligase master mix
  • Amount11-x µL nuclease-free H2O

Mix by pipetting and spin down.
Temperature25 °C Duration00:20:00




20m
Add Amount25 µL TE(pH8.0).
The cDNA is purified using ReagentAgencourt AMPure XPBeckman CoulterCatalog #A63880 .

Add Amount40 µL (X0.8 volume) AMPure XP reagent and mix by pipetting.
Incubate on a rotor mixer.
Duration00:05:00 TemperatureRoom temperature
Spin down and pellet on a magnet. wait for Duration00:01:00 .
Pipette off the supernatant.
Wash twice by Amount200 µL 70 % ethanol and remove the ethanol using a pipette and discard.
Spin down and pipette off any residual ethanol.
Resuspend pellet in Amount20 µL nuclease-free water.
Temperature37 °C Duration00:03:00 and tapping occasionally.
Incubate on a rotor mixer.
Duration00:07:00
Spin down and pellet the beads on the magnet until the elute is clear and colourless.
Remove retain Amount20 µL elute into a new tube.
DNA concentration is measured using a Qubit 4 Fluorometer with ReagentQubit 1X dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q33230 .

  • Amount199 µL 1X working solution
  • Amount1 µL DNA

Mix by vortexing.

Incubate Duration00:02:00 TemperatureRoom temperature and measure.
Convert nanogram(ng) into femtomole(fmol) by a calculator.
Note
The molar concentration of the cDNA sample can be converted based on the length of the major band confirmed by electrophoresis after T7 endonuclease treatment. Typically, the fragment lengths are around 2000 bases pairs.

Adaptor ligation
Adaptor ligation
20m
20m
Pool each barcoded sample into a 0.2ml PCR tube (Total 100–200 fmol).
Adaptor Ligation with pooled samples is performed using
Ligation Sequencing Kit - Oxford Nanopore Technologies Catalog #SQK-LSK109, Native Barcoding Expansion - Oxford Nanopore Technologies Catalog #EXP-NBD104, #EXP-NBD114 and
ReagentNEBNext Quick Ligation Module - 20 rxnsNew England BiolabsCatalog #E6056S .

Total 50 μl reaction

  • Amountx µL DNA (100-200 fmol)
  • Amount2.5 µL Adaptor Mix II
  • Amount10 µL NEB Next Quick Ligation Reaction Buffer(5X)
  • Amount5 µL Quick T4 DNA ligase
  • Amount32.5-x µL nuclease-free H2O

mix gently and incubate.
Temperature25 °C Duration00:20:00
20m
The adaptor-ligated cDNA is purified using ReagentAgencourt AMPure XPBeckman CoulterCatalog #A63880 .
Prepare AMpure XP beads for use; resuspend by vortexing.
Transfer amplified DNA sample to 1.5ml low binding tube.

Add Amount80 µL AMPure XP reagent and mix by pipetting.
Incubate on a rotor mixer.
Duration00:05:00 TemperatureRoom temperature
Spin down and pellet on a magnet. Wait for Duration00:01:00 and pipette off the supernatant.
  • Wash twice by Amount200 µL . Short Fragment Buffer(SFB) and remove the ethanol using a pipette and discard.
Spin down and pipette off any residual SFB.
  • Resuspend pellet in Amount12 µL Elution Buffer (EB)
Temperature37 °C Duration00:03:00 and tapping occasionally.
Incubate on a rotor mixer.
Duration00:07:00
Spin down and pellet the beads on the magnet until the elute is clear and colourless.
Remove retain Amount12 µL elute into a new tube.
DNA concentration is measured using a Qubit 4 Fluorometer with ReagentQubit 1X dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q33230 .

  • Amount199 µL 1X working solution
  • Amount1 µL DNA

Mix by vortexing.

Incubate Duration00:02:00 TemperatureRoom temperature and measure.

Sequencing by MinION
Sequencing by MinION
Sequencing according to the manufacturer's instructions.