Direct cDNA Sequencing (SQK-DCS109)

Prepare the RNA in Nuclease-free water.

Transfer 100ng into a 1.5 ml Eppendorf DNA LoBind tube.

Adjust the volume to up to 7.5µL with Nuclease-free water.

Mix by flicking the tube to avoid unwanted shearing.

Spin down briefly in a microfuge.

Prepare the following reaction in a 0.2 ml PCR tube:

• x μl poly A+ RNA, 100 ng
• 2.5µL
• 1µL
• 7.5-x μl RNase-free water

Mix gently by flicking the tube, and spin down.

Incubate at 65°C for 0h 5m 0s and then snap cool on a pre-chilled freezer block.

In a separate tube, mix together the following:

• 4µL
• 1µL
• 1µL
• 2µL

Mix gently by flicking the tube, and spin down.

Add the strand-switching buffer to the snap-cooled, annealed mRNA, mix by flicking the tube and spin down.

Incubate at 42°C for 0h 2m 0s.

Add 1µL. The total volume is now 20µL.

Mix gently by flicking the tube, and spin down.

Incubate using the following protocol:

• Reverse transcription and strand-switching 1h 30m 0s @ 42°C (1 cycle)
• Heat inactivation 0h 5m 0s @ 85°C (1 cycle)
• Hold @ 4°C

Add 1µL to the reverse transcription reaction.

Incubate the reaction for 0h 10m 0s at 37°C.

Resuspend the AMPure XP beads by vortexing.

Transfer the sample to a clean 1.5 ml Eppendorf DNA LoBind tube.

Add 17µL to the reaction and mix by flicking the tube.

Incubate on a Hula mixer (rotator mixer) for 0h 5m 0s at 37Room temperature.

Prepare 500µL fresh 70%ethanol in Nuclease-free water.

Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant.

Keep the tube on the magnet and wash the beads with 200µL without disturbing the pellet. Remove the ethanol using a pipette and discard.

Repeat the previous step.

Spin down and place the tube back on the magnet. Pipette off any residual ethanol. Allow to dry for ~0h 0m 30s, but do not dry the pellet to the point of cracking.

Remove the tube from the magnetic rack and resuspend pellet in 20µL.

Incubate on a Hula mixer (rotator mixer) for 0h 10m 0s at 37Room temperature.

Pellet beads on magnet until the eluate is clear and colourless.

Remove and retain 20µL into a clean 1.5 ml Eppendorf DNA LoBind tube.

Prepare the following reaction in a 0.2 ml thin-walled PCR tube:

• 25µL
• 2µL
• 20µL
• 3µL

Incubate using the following protocol:

• 94°C 0h 1m 0s 1
• 50°C 0h 1m 0s 1
• 65°C 0h 15m 0s 1
• 4°C ∞

Resuspend the AMPure XP beads by vortexing.

Transfer the sample to a clean 1.5 ml Eppendorf DNA LoBind tube.

Add 40µL to the reaction and mix by flicking the tube.

Incubate on a Hula mixer (rotator mixer) for 0h 5m 0s at 4Room temperature.

Prepare 500µL.

Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant.

Keep the tube on the magnet and wash the beads with 200µL without disturbing the pellet. Remove the ethanol using a pipette and discard.

Repeat the previous step.

Spin down and place the tube back on the magnet. Pipette off any residual ethanol. Allow to dry for ~0h 0m 30s, but do not dry the pellet to the point of cracking.

Remove the tube from the magnetic rack and resuspend pellet in 21µL.

Incubate on a Hula mixer (rotator mixer) for 0h 10m 0s at 4Room temperature.

Pellet beads on magnet until the eluate is clear and colourless.

Remove and retain 21µL into a clean 1.5 ml Eppendorf DNA LoBind tube.

Analyse 1µL for size, quantity and quality.

Perform end repair and dA-tailing of fragmented DNA as follows:

• 20µL
• 30µL
• 7µL
• 3µL

Mix gently by pipetting and spin down.

Using a thermal cycler, incubate at 20°C for 0h 5m 0s and 65°C for 0h 5m 0s.

Resuspend the AMPure XP beads by vortexing.

Transfer the sample to a 1.5 ml DNA LoBind Eppendorf tube.

Add 60µL to the end-prep reaction and mix by pipetting.

Incubate on a Hula mixer (rotator mixer) for 0h 5m 0s at 65Room temperature.

Prepare 500µL.

Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant.

Keep the tube on the magnet and wash the beads with 200µL without disturbing the pellet. Remove the ethanol using a pipette and discard.

Repeat the previous step.

Spin down and place the tube back on the magnet. Pipette off any residual ethanol. Allow to dry for ~0h 0m 30s, but do not dry the pellet to the point of cracking.

Remove the tube from the magnetic rack and resuspend pellet in 30µL. Incubate for 0h 2m 0s at 65Room temperature.

Pellet the beads on a magnet until the eluate is clear and colourless.

Remove and retain 30µL into a clean 1.5 ml Eppendorf DNA LoBind tube.

Take forward 30µL into adapter ligation.

Check the contents of each tube are clear of any precipitate and are thoroughly mixed before setting up the reaction.

Mix the contents of each tube by flicking.

Check that there is no precipitate present (DTT in the Blunt/TA Master Mix can sometimes form a precipitate).

Spin down briefly before accurately pipetting the contents in the reaction.

Taking the end-prepped DNA, perform adapter ligation as follows, mixing by flicking the tube between each sequential addition.

• 30µL
• 5µL
• 50µL
• 15µL

Mix gently by flicking the tube, and spin down.

Incubate the reaction for 0h 10m 0s at 65Room temperature.

Resuspend the AMPure XP beads by vortexing.

Add 40µL to the adapter ligation reaction from the previous step and mix by pipetting.

Incubate on a Hula mixer (rotator mixer) for 0h 5m 0s at 65Room temperature.

Place on magnetic rack, allow beads to pellet and pipette off supernatant.

Add 200µL to the beads. Resuspend the beads by pipetting up and down. Return the tube to the magnetic rack, allow beads to pellet and pipette off the supernatant.

Repeat the previous step.

Spin down and place the tube back on the magnet. Pipette off any residual supernatant.

Remove the tube from the magnetic rack and resuspend pellet in 25µL.

Incubate on a Hula mixer (rotator mixer) for 0h 10m 0s at 65Room temperature.

Pellet the beads on a magnet until the eluate is clear and colourless.

Remove and retain 25µL into a clean 1.5 ml Eppendorf DNA LoBind tube.

Quantify 1µL using a Qubit fluorometer - recovery aim ~60 fmol.

Thaw the Sequencing Buffer (SQB), Loading Beads (LB), Flush Tether (FLT) and one tube of Flush Buffer (FB) at 65Room temperature.

Mix the Sequencing Buffer (SQB), Flush Buffer (FB) and Flush Tether (FLT) tubes by vortexing and spin down at 65Room temperature.

To prepare the flow cell priming mix, add 30µL directly to 1 tube of thawed and mixed Flush Buffer (FB), and mix by vortexing.

Load the flow cell(s) into the docking ports within the PromethION.

Prime the flow cell using the following steps, taking care to avoid the introduction of air bubbles.

Turn the valve to expose the inlet port (Port 1).

A small tract of air will be visible beyond the inlet port. Draw back a small volume to remove any air bubbles (a few µls):

a. Set a P1000 pipette to 200 µl

b. Insert the tip into the inlet port

c. Turn the wheel until the dial shows 220-230 µl, or until you can see a small volume of buffer entering the pipette tip.

Using a P1000 pipette, flush 500µL into the inlet port of the flow cell, avoiding the introduction of air bubbles.

Wait 0h 5m 0s. During this time you can prepare your library for loading, as described in the next steps.

Repeat the priming step with another 500µL.

Thoroughly mix the contents of the Loading Beads (LB) tubes by vortexing.

In a new tube, prepare the library for loading as follows:

• 75µL
• 51µL
• 24µL

Load your sample.

Load 150µL through the inlet port.

Close the valve to seal the inlet port and close the PromethION lid when ready.

Wait a minimum of 0h 10m 0s after loading the flow cells onto the PromethION before initiating any experiments. This will help to increase the sequencing output.

After your sequencing experiment is complete, if you would like to reuse the flow cell, please follow the Wash Kit instructions and store the washed flow cell at 2°C - 8°C, OR

Follow the returns procedure by washing out the flow cell ready to send back to Oxford Nanopore.