Editing Nanopore RNA Sequencing Protocol (section)

====SpotON Flow Cell====

Below is a list of materials and reagents needed for the loading process

{| class="wikitable"
|+Flow Cell Loading Materials
|-
! scope="col"| Type
! scope="col"| Items
|-
|'''Materials'''
|
* Prepared RNA library (see previous step).
* RNA Running Buffer (RRB, '''Red Cap''').
* Flow Cell Priming Kit (EXP-FLP002).
|-
|'''Consumables'''
|
* MinION Mk1B.
* SpotON Flow Cell.
* Nuclease-free water (e.g., ThermoFisher, AM9937).
* 1.5 mL Eppendorf DNA LoBind tubes.
|}

# '''Thaw the RNA Running Buffer (RRB), Flush Tether (FLT) and one tube of Flush Buffer (FB) at room temperature.''' This should be done already if you followed the tips from the previous section.
# '''Mix the RNA Running Buffer (RRB), Flush Buffer (FB) and Flush Tether (FLT) tubes thoroughly by vortexing and spin down at room temperature.'''
# '''To prepare the flow cell priming mix, add 30 μL of thawed and mixed Flush Tether (FLT) directly to the tube of thawed and mixed Flush Buffer (FB), and mix by vortexing at room temperature.''' This may already be done from a previous run.
# '''Open the MinION device lid and slide the flow cell under the clip.''' Press down firmly on the flow cell to ensure correct thermal and electrical contact.
# '''(Optional) On the MinKNOW software, run a flow cell check to determine if the flow cell is below warranty.'''
# '''Slide the priming port cover clockwise to open the priming port.'''

{{warning|Take care when drawing back buffer from the flow cell. Do not remove more than 20-30 μL, and make sure that the array of pores are covered by buffer at all times. Introducing air bubbles into the array can irreversibly damage pores.}}

<ol start="7">
<li>'''After opening the priming port, check for a small air bubble under the cover. Draw back a small volume to remove any bubbles:'''</li>
<ol>
<li>Set a P1000 pipette to 200 μL.</li>
<li>Insert the tip into the priming port.</li>
<li>Turn the wheel until the dial shows 220-230 μL, to draw back 20-30 μL, or until you can see a small volume of buffer entering the pipette tip. Visually check that there is continuous buffer from the priming port across the sensor array.</li>
</ol>
<li>'''Load 800 μL of the priming mix into the flow cell via the priming port, avoiding the introduction of air bubbles. Wait for five minutes. During this time, prepare the library for loading by following the steps below.'''</li>
</ol>

{{warning|Thoroughly mix the contents of the RRB tube by vortexing or pipetting, and spin down briefly.}}

<ol start="9">
<li>'''Take 20 μL of the prepared RNA library and mix it with 17.5 μL of nuclease-free water.'''</li>
<li>'''In a new tube, prepare the library for loading as follows:'''</li>
</ol>

{| class="wikitable"
|+RNA Sample Preparation
|-
! scope="col"| Reagent
! scope="col"| Volume per flow cell
|-
|RNA Running Buffer (RRB, '''Red Cap''')
|37.5 μL
|-
|RNA library in nuclease-free water
|37.5 μL
|-
|'''Total'''
|'''75 μL'''
|}

<ol start="11">
<li>'''Complete the flow cell priming:'''</li>
<ol>
<li>Gently lift the SpotON sample port cover to make the SpotON sample port accessible.</li>
<li>'''Load 200 μL''' of the priming mix into the flow cell priming port (not the SpotON sample port), avoiding the introduction of air bubbles.</li>
</ol>
<li>'''Mix the prepared library gently by pipetting up and down just prior to loading.'''</li>
<li>'''Add 75 μL of sample to the Flow Cell via the SpotON sample port in a dropwise fashion. Ensure each drop flows into the port before adding the next.'''</li>
<li>'''Gently replace the SpotON sample port cover, making sure the bung enters the SpotON port, close the priming port and replace the MinION device lid.'''</li>
</ol>

Begin data acquisition and basecalling in the ''MinKNOW'' software.