Editing Nanopore RNA Sequencing Protocol (section)

===RNA Library Preparation===

{| class="wikitable"
|+Materials and Equipment
|-
! scope="col"| Type
! scope="col"| Items
|-
|'''Materials'''
|
* 50 ng of poly(A)-tailed RNA or 500 ng of total RNA in 9 μL. '''Note:''' For my best run, it was 333 ng in 9 μL of pure mRNA.
* RT Adapter (RTA, '''Blue Cap''').
* RNA CS (RCS, '''Yellow Cap''').
* RNA Adapter (RMX, '''Green Cap''').
* Wash Buffer (WSB, '''Orange Cap''').
* Elution Buffer (ELB, '''Black Cap''').
|-
|'''Consumables'''
|
* NEBNext® Quick Ligation Reaction Buffer (NEB, B6058).
* T4 DNA Ligase 2M U/mL (NEB, M0202T/M).
* 0.2 mL thin-walled PCR tubes.
* Nuclease-free water (e.g. ThermoFisher, AM9937).
* Agencourt RNAClean XP beads (Beckman Coulter™, A63987).
* Freshly prepared 70% ethanol in nuclease-free water.
* 1.5 mL Eppendorf DNA LoBind tubes.
* SuperScript III Reverse Transcriptase (Thermo Fisher Scientific, 18080044).
* 10 mM dNTP solution (e.g. NEB N0447).
* Qubit dsDNA HS Assay Kit (ThermoFisher, cat # Q32851).
|-
|'''Equipment'''
|
* Magnetic separator, suitable for 1.5 mL Eppendorf tubes.
* Hula mixer (gentle rotator mixer).
* Termal cycler.
|-
|'''Optional Equipment'''
|
* Qubit fluorometer (or equivalent for QC check).
|}

{{warning|'''You need to test your mRNA purity before proceeding'''. This can be done using the Nanodrop. You should have a decent concentration with 260/230 and 260/280 ratios of 2.0 or better. A low 260/230 ratio indicates organics in your sample, which can damage the flow cell pores, whereas a low 260/280 ratio indicates proteins, which can clog them. Finally, you may want to dilute your input sample so that you're loading your ideal amount in 9 μL of sample. More info on interpreting Nanodrop results can be found [https://dnatech.genomecenter.ucdavis.edu/wp-content/uploads/2016/03/InterpretingSpectrometry.pdf here].}}

# '''Prepare the RNA in nuclease-free water.'''
#* Transfer '''50 ng†''' of poly(A)-tailed RNA or '''500 ng''' of total RNA into a 1.5 mL Eppendorf DNA LoBind tube.
#* Adjust† the volume to 9 μL with nuclease-free water.
#* Mix thoroughly by flicking the tube to avoid unwanted shearing.
#* Spin down briefly in a microfuge. 
†Note: For my best run, I diluted the sample and loaded 9 μL of pure mRNA directly. This was around 333 ng total in 9 μL (~37 ng/μL).

{{warning|'''Only poly(A)-tailed RNA will eventually be read by the nanopore system'''. Therefore, if you are interested in non-poly(A)-tailed RNA (such as long non-coding RNA), you will need to poly(A)-tail it separately and purify it prior to the run. Additionally, '''the old protocol called for 500 ng of poly(A)-tailed RNA''' instead of 50 ng (it is unclear at the present on why this was changed). My best run varied from both of these amounts. Finally, '''according to ONT, there is no difference in using total or mRNA for human samples''', however, they acknowledge that this is not the case for other species (such as yeast) and that some users have reported better results with purified mRNA across species.}}

<ol start="2">
<li>'''In a 0.2 mL thin-walled PCR tube, mix the reagents in the following order:'''</li>
</ol>

{| class="wikitable"
|+Prepping Sample for Adapter Ligation
|-
! scope="col"| Reagent
! scope="col"| Volume
|-
|NEBNext Quick Ligation Reaction Buffer
|3.0 μL
|-
|RNA Sample
|9.0 μL
|-
|RNA CS (RCS, '''Yellow Cap'''), 110 nM
|0.5 μL
|-
|RT Adapter (RTA, '''Blue Cap''')
|1.0 μL
|-
|T4 DNA Ligase
|1.5 μL
|-
|'''Total'''
|'''15 μL'''
|}

<ol start="3">
<li>'''Mix by pipetting and spin down.'''</li>
<li>'''Incubate the reaction for 10 minutes at room temperature.'''</li>
<li>'''Mix the following reagents together to make the reverse transcription master mix:'''</li>
</ol>

{| class="wikitable"
|+Reverse Transcription Master Mix
|-
! scope="col"| Reagent
! scope="col"| Volume
|-
|Nuclease-free water
|9.0 μL
|-
|10 mM dNTPs
|2.0 μL
|-
|5x first-strand buffer
|8.0 μL
|-
|0.1 M DTT
|4.0 μL
|-
|'''Total'''
|'''23.0 μL'''
|}

<ol start="6">
<li>'''Add the master mix to the 0.2 mL PCR tube containing the RT adapter-ligated RNA from the "RT Adapter ligation" step. Mix by pipetting.'''</li>
<li>'''Add 2 μL of SuperScript III Reverse Transcriptase to the reaction and mix by pipetting.'''</li>
<li>'''Place the tube in a thermal cycler and incubate at 50°C for 50 minutes, then 70°C for 10 minutes, and bring the sample to 4°C before proceeding to the next step.'''</li>
</ol>

{{note|This can be found on the thermal cycler near Tie's bench as the program '''RT Ligation Nanopore'''. This is also the point where you can take a break, since this step will take approximately 1 hour and 6 minutes to complete.}}

<ol start="9">
<li></li>
</ol>

<ol start="9">
<li>'''Transfer the sample to a clean 1.5 mL Eppendorf DNA LoBind tube.'''</li>
<li>'''Resuspend the stock of Agencourt RNAClean XP beads by vortexing.'''</li>
<li>'''Add 72 μL of resuspended Agencourt RNAClean XP beads to the reverse transcription reaction and mix by pipetting.'''</li>
<li>'''Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.'''</li>
<li>'''Prepare 200 μL of fresh 70% ethanol in nuclease-free water (i.e., mix 140 μL pure ethanol with 60 μL PCR water in a clean Eppendorf tube).'''</li>
<li>'''Spin down and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant (use a p100 if possible to avoid pulling in RNA beads).'''</li></ol>
{{note|For the spin down, I used 2,500g for 1 minute.}}
<ol start="9">
<li>'''Keep the tube on magnet, and wash the beads with 150 μL of freshly prepared 70% ethanol without disturbing the pellet as described below.'''</li>
{{tip|Rotate the tube ''very fast'' to avoid the pellet from smearing. You want it to migrate directly across the tube through the ethanol, but otherwise remain intact.}}
<ul>
<li>Keeping the magnetic rack on the benchtop, rotate the bead-containing tube by 180°. Wait for the beads to migrate towards the magnet and form a pellet. Wait 2.5 minutes.</li>
<li>Rotate the tube 180° again (back to the starting position), and wait for the beads to pellet. Wait 2.5 minutes.</li>
</ul>
<li>'''Remove the 70% ethanol using a pipette and discard.'''</li>
<li>'''Spin down and place the tube back on the magnet until the eluate is clear and colourless. Keep the tubes on the magnet and pipette off any residual ethanol.'''</li>
</ol>

{{note|For the spin down, I used 2,500g for 1 minute.}}

<ol start="18">
<li>'''Remove the tube from the magnetic rack and resuspend pellet (gentle pipetting) in 20 μL nuclease-free water. Incubate for 5 minutes at room temperature.'''</li>
<li>'''Pellet the beads on a magnet until the eluate is clear and colourless.'''</li>
<li>'''Remove and retain 20 μL of eluate into a clean 1.5 mL Eppendorf DNA LoBind tube.'''</li>
<li>'''In the same 1.5 mL Eppendorf DNA LoBind tube, mix the reagents in the following order:'''</li>
</ol>

{| class="wikitable"
|+RNA Adapter Mix
|-
! scope="col"| Reagent
! scope="col"| Volume
|-
|NEBNext Quick Ligation Reaction Buffer
|8.0 μL
|-
|RNA Adapter (RMX, '''Green Cap''')
|6.0 μL
|-
|Nuclease-free water
|3.0 μL
|-
|T4 DNA Ligase
|3.0 μL
|-
|'''Total (including all reagents)'''
|'''40 μL'''
|}

<ol start="22">
<li>'''Mix by pipetting.'''</li>
<li>'''Incubate the reaction for 10 minutes at room temperature.'''</li>
<li>'''Resuspend the stock of Agencourt RNAClean XP beads by vortexing.'''</li>
<li>'''Add 16 μL of resuspended Agencourt RNAClean XP beads to the reaction and mix by pipetting.'''</li>
</ol>

{{tip|Check to ensure that the '''Elution Buffer''' is thawing at this point. In my experience, even on ice it tends to remain frozen.}}

<ol start="26">
<li>'''Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.'''</li>
<li>'''Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant.'''</li>
</ol>

{{note|For the spin down, I used '''3,000g''' for 1 minute (note the change!).}}
{{tip|Start thawing the '''Flush Buffer''', '''Flush Tether''', and '''RNA Running Buffer''' at room temperature in preparation for flow cell priming and loading. If you are loading using the '''Flongle''' reagents, also set the '''Sequencing Buffer''', '''Flongle Flush Buffer''', and '''Loading Beads II''' out to get to room temperature.}}

<ol start="28">
<li>'''Add 150 μL of the Wash Buffer (WSB) to the beads. Close the tube lid and resuspend the beads by flicking the tube. Return the tube to the magnetic rack, allow the beads to pellet and pipette off the supernatant.'''</li>
<li>'''Repeat the previous step.'''</li>
</ol>

{{warning|Agitating the beads results in a more efficient removal of free adapter, compared to adding the wash buffer and immediately aspirating.}}

{{tip|'''Make sure''' the '''Elution Buffer''' is completely thawed at this point.}}

<ol start="30">
<li>'''Remove the tube from the magnetic rack and resuspend pellet in 21 μL Elution Buffer by the gently flicking the tube and pipetting. Incubate for 10 minutes at room temperature.'''</li>
<li>'''Pellet the beads on a magnet until the eluate is clear and colourless.'''</li>
<li>'''Repeat the above two steps to increase your yield using the same 21 μL of Elution Buffer. Mix by pipetting and incubate for 10 minutes prior to pelleting on magnet.'''</li>
<li>'''Remove and retain 21 μL of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.'''</li>
</ol>

Quantify 1 μL of reverse-transcribed and adapted RNA using the Qubit fluorometer DNA HS assay - recovery aim ~20 ng. If you have omitted the reverse transcription step, please use the Qubit RNA HS Assay Kit instead. However, please note that the kit will measure all RNA present, including any non-adapted RNA that has been carried through in the RNAClean XP bead clean-up. The reported quantity of RNA may therefore not fully represent the amount of sequenceable RNA.

Note that you can view the concentration of your library on the Nanodrop and may or may not get a reading. For a good run, I recorded a slight "hill" peak around 260 nm and around 2.6 ng/μL final concentration. '''Since your library is double stranded, use the DNA setting on the nanodrop'''. If you use RNA, you will get an inaccurate reading. 

Note that for the loading step, you will want to load the following amounts of RNA for a good sequencing run:

{| class="wikitable"
|+RNA Loading Amounts
|-
! scope="col"| Flow Cell
! scope="col"| Amount of mRNA (in fmol)
! scope="col"| Amount in ng (if N50 = 1,400 bases)
|-
|'''Flongle'''
|3-20 fmol; 10-15 fmol ideal
|~ 6.751 ng (my best run loaded 2.6 ng/μL in 7 μL, for 18.2 ng total)
|-
|'''Standard MinION'''
|44.44 fmol
|20 ng
|}

You can also calculate your own loading amounts using the [https://nebiocalculator.neb.com/#!/ssrnaamt NEBio RNA mass calculator] if your RNA is of a different length.