Editing Nanopore RNA Sequencing Protocol (section)

====Flongle 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).
* Flongle Sequencing Expansion (EXP-FSE001).
* Flow Cell Priming Kit (EXP-FLP002).
|-
|'''Consumables'''
|
* MinION Mk1B.
* Flongle Adapter.
* Flongle Flow Cell.
* Nuclease-free water (e.g., ThermoFisher, AM9937).
* 1.5 mL Eppendorf DNA LoBind tubes.
|}

# '''Thaw the Sequencing Buffer (SB), Loading Beads II (LBII), Flow Cell Tether (FCT, found in Priming Kit), and Flush Buffer (FB, use the one from the Flongle kit) at room temperature'''. This should already be done if you followed the tips from the previous section.
# '''Mix the SB, LBII, FCT, and FB by vortexing and spin down at room temperature'''.
# '''Prepare the priming mix as follows:'''

{| class="wikitable"
|+Priming Mix
|-
! scope="col"| Reagent
! scope="col"| Volume
|-
|Flush Buffer
|117 μL
|-
|Flow Cell Tether
|3 μL
|-
|'''Total'''
|'''120 μL'''
|}

<ol start="4">
<li>'''Place the Flongle Flow Cell into the adapter in the MinION. Peel back the seal from the Flongle flow cell carefully until the sample port is exposed. Tape down the adhesive onto the lid of the MinION to keep it held back.'''</li>
<li>'''Using a 200 μL pipette, draw 100 μL of your priming mix into the tip. Ensure that no air is in the bottom of the tip past the solution.'''</li>
<li>'''Place the tip of the pipette securely into the sample port. Turn the dial of the pipette counter clockwise a time or two and you should see a small amount of yellow-green fluid come into the tip.''' This helps to ensure there are no air bubbles present.</li>
<li>'''Once confirmed, slowly dispense the priming fluid into the flow cell by slowly turning the dial of the pipette clockwise (towards 0). Do not push down on the plunger to eject the fluid.''' You should see some fluid being to enter the waste port.</li>
<li>'''Remove the pipette tip just before the fluid reaches the bottom.'''</li>
<li>In a new 1.5 μL tube, add the reagents below in the order they are listed to make the RNA sample solution:</li>
</ol>

{{warning|The '''Loading Beads II''' tend to settle quickly, so you will need to vortex it again before drawing it to make the solution.}}

{| class="wikitable"
|+RNA Sample Preparation
|-
! scope="col"| Reagent
! scope="col"| Volume
|-
|Sequencing Buffer (SB)
|15 μL
|-
|Loading Beads II (LBII)
|10 μL
|-
|3-20 fmol mRNA Library
|5 μL
|-
|'''Total'''
|'''30 μL'''
|}

<ol start="10">
<li>'''Mix the solution by gently pipetting up and down 10-20 times.</li>
<li>'''Draw the entire volume into the pipette. Make sure there is no air at the tip of the pipette.'''</li>
<li>'''Insert the tip of the pipette into the sample port and slowly dispense the library into the flow cell by slowly turning the dial of the pipette clockwise. Do not push down on the plunger to eject the fluid.'''</li>
<li>'''Remove the pipette tip just before you finish adding all of the solution to avoid the introduction of air bubbles.'''</li>
<li>'''Using a clean pipette tip, dropwise add the remaining priming solution to top off the sample port tip.'''</li>
<li>'''Seal the Flongle flow cell by attaching the adhesive tape back over the top of the flow cell.''' Make sure the back of the tape covers the waste ports on the left and right sides of the Flongle, and seal with ONE stroke over the top. Close the MinION lid.</li>
</ol>

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

{{warning|'''VERY IMPORTANT:''' It is highly advised that you start ''Sequencing'' on your flow cell before you prime the flow cell or load your library. This will allow you to see the actual pore availability prior to loading your sample and whether the flow cell is in decent shape. If there is an issue during sequencing, this will also allow you to easily determine whether the problem is due to the flow cell or due to your sample preparation/loading. If the flow cell does not have a large number of "available" pores at the start of the run, '''DO NOT LOAD YOUR SAMPLE''' and contact ''ONT'' for a replacement flow cell.

In addition, on '''Flongles you must set the active channel selection (MUX Scans) interval to longer than your experiment''', i.e., to 24 hours or more. This is because MUX Scans can damage Flongle pores for some unknown reason. '''Make sure you set the interval to longer than the run, rather than disable active channel selection''', because I have found it will still continually scan pores with active channel selection disabled.}}