Direct cDNA sequencing V14 with SQK-LSK114

This protocol describes how to carry out sequencing of cDNA using a reverse transcription and strand-switching method and the Ligation Sequencing Kit V14 (SQK-LSK114) This protocol is recommended for users who: - Are interested in exploring novel RNA biology. - Are looking for splice variant and fusion transcript analysis. - Do not wish to use PCR. - Wish to preserve quantitative information in samples likely to be impacted by PCR bias. - Would like full-length cDNA strands. - Want to achieve median raw read accuracy of Q20+ (99%) and above. - Want to optimise their sequencing experiment for output.

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Version 1 Current

Effective: 2025-11-04

First version.

Procedure Steps (Version 1)

Thaw the following reagents and spin down briefly using a microfuge, before mixing as indicated in the table below, and place on ice.

Reagent 1. Thaw at room temperature 2. Briefly spin down 3. Mix well by pipetting User-supplied VN Primer diluted to 2 µM ✓ ✓ ✓ User-supplied Strand-Switching Primer diluted to 10 µM ✓ ✓ ✓ 10 mM dNTP solution ✓ ✓ ✓ RNaseOUT Not frozen ✓ ✓ Maxima H Minus Reverse Transcriptase Not frozen ✓ ✓ Maxima H Minus 5x RT Buffer ✓ ✓ Mix by vortexing

Prepare the RNA in nuclease-free water

Transfer 100 ng Poly(A)+ RNA or 1 μg of total RNA into a 0.2 ml PCR tube
Adjust the volume to up to 7.5 μl with nuclease-free water
Mix by flicking the tube to avoid unwanted shearing
Spin down briefly in a microfuge

Prepare the following reaction in the 0.2 ml PCR tube containing the prepared RNA input:

Reagent Volume RNA input (100 ng Poly(A)+ RNA or 1 μg of total RNA) from step above 7.5 μl VN Primer diluted to 2 μM 2.5 μl 10 mM dNTPs 1 μl Total volume 11 μl

Mix gently by flicking the tube, and spin down.

Incubate at 65°C for 5 minutes and then snap cool on a pre-chilled freezer block for 1 minute.

In a separate tube, mix together the following:

Reagent Volume 5x RT Buffer 4 μl RNaseOUT 1 μl Nuclease-free water 1 μl Strand-Switching Primer diluted to 10 µM 2 μl Total 8 μl

Mix gently by flicking the tube, and spin down.

Add the 8 μl of strand-switching reagents (prepared in steps 6-7) to the 11 μl of snap-cooled mRNA (from steps 2-5). Mix by flicking the tube and spin down.

Incubate at 42°C for 2 minutes in the thermal cycler.

Add 1 µl of Maxima H Minus Reverse Transcriptase. The total volume is now 20 µl.

Mix gently by flicking the tube, and spin down.

Incubate using the following protocol using a thermal cycler:

Cycle step Temperature Time No. of cycles Reverse transcription and strand-switching 42°C 90 mins 1 Heat inactivation 85°C 5 mins 1 Hold 4°C ∞

Thaw the following reagents and spin down briefly using a microfuge, before mixing as indicated in the table below, and place on ice.

Reagent 1. Thaw at room temperature 2. Briefly spin down 3. Mix well by pipetting User-supplied PR2 Primer diluted to 10 µM ✓ ✓ ✓ RNase Cocktail Enzyme Mix Not frozen ✓ ✓ LongAmp Taq 2X Master Mix ✓ ✓ ✓

Thaw the AMPure XP Beads (AXP) at room temperature and mix by vortexing. Keep the beads at room temperature.

Add 1 µl RNase Cocktail Enzyme Mix (ThermoFisher, cat # AM2286) to the reverse transcription reaction.

Incubate the reaction for 10 minutes at 37° C in a thermal cycler.

Resuspend the AMPure XP beads (AXP) by vortexing.

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

Add 17 µl of resuspended AMPure XP beads (AXP) to the reaction and mix by flicking the tube.

Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.

Prepare 500 μl of fresh 80% 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 tubes on the magnet and wash the beads with 200 µl of freshly prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard. If the pellet was disturbed, wait for beads to pellet again before removing the ethanol.

Repeat the previous step.

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

Remove the tube from the magnetic rack and resuspend pellet in 20 µl nuclease-free water.

Incubate on a Hula mixer (rotator mixer) for 10 minutes at room temperature.

Briefly spin down the tube and pellet the beads on the magnet until the eluate is clear and colourless, for at least 1 minute.

Remove and retain 20 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.

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

Reagent Volume 2x LongAmp Taq Master Mix 25 μl PR2 Primer diluted to 10 μM 2 μl Reverse-transcribed sample from above 20 μl Nuclease-free water 3 μl Total 50 μl

Incubate using the following protocol:

Cycle step Temperature Time No. of cycles Denaturation 94 °C 1 mins 1 Annealing 50 °C 1 mins 1 Extension 65 °C 15 mins 1 Hold 4 °C ∞

Resuspend the AMPure XP beads (AXP) by vortexing.

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

Add 40 µl of resuspended AMPure XP beads (AXP) to the reaction and mix by flicking the tube.

Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.

Prepare 500 μl of fresh 80% 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 tubes on the magnet and wash the beads with 200 µl of freshly prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.

If the pellet was disturbed, wait for beads to pellet again before removing the ethanol.

Repeat the previous step.

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

Remove the tube from the magnetic rack and resuspend pellet in 21 µl nuclease-free water.

Incubate on a Hula mixer (rotator mixer) for 10 minutes at room temperature.

Briefly spin down the tube and pellet the beads on the magnet until the eluate is clear and colourless, for at least 1 minute.

Remove and retain 21 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.

Analyse 1 µl of the strand-switched DNA for size, quantity and quality using an Agilent Bioanalyzer and Qubit fluorometer (or equivalent).

Prepare the NEBNext Ultra II End Repair / dA-tailing Module reagents in accordance with manufacturer's instructions, and place on ice:

For optimal perfomance, NEB recommend the following:

Thaw all reagents on ice.
Flick and/or invert the reagent tubes to ensure they are well mixed. Note: Do not vortex the Ultra II End Prep Enzyme Mix.
Always spin down tubes before opening for the first time each day.
The Ultra II End Prep Buffer may have a little precipitate. Allow the mixture to come to room temperature and pipette the buffer up and down several times to break up the precipitate, followed by vortexing the tube for 30 seconds to solubilise any precipitate.

Combine the following reagents in a 0.2 ml PCR tube:

Reagent Volume cDNA sample 20 µl Nuclease-free water 30 µl Ultra II End-prep reaction buffer 7 µl Ultra II End-prep enzyme mix 3 µl Total 60 µl

Thoroughly mix the reaction by gently pipetting and briefly spinning down.

Using a thermal cycler, incubate at 20°C for 5 minutes and 65°C for 5 minutes.

Resuspend the AMPure XP Beads (AXP) by vortexing.

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

Add 60 µl of resuspended the AMPure XP Beads (AXP) to the end-prep reaction and mix by flicking the tube.

Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.

Prepare 500 μl of fresh 80% ethanol in nuclease-free water.

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

Keep the tube on the magnet and wash the beads with 200 µl of freshly prepared 80% ethanol 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 ~30 seconds, but do not dry the pellet to the point of cracking.

Remove the tube from the magnetic rack and resuspend pellet in 61 µl nuclease-free water. Incubate for 2 minutes at room temperature.

Pellet the beads on a magnet until the eluate is clear and colourless, for at least 1 minute.

Remove and retain 61 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.

Quantify 1 µl of eluted sample using a Qubit fluorometer.

Take forward the 60 µl of repaired and end-prepped cDNA into the adapter ligation step. However, at this point it is also possible to store the sample at 4°C overnight.

Spin down the Ligation Adapter (LA) and Quick T4 Ligase, and place on ice.

Thaw Ligation Buffer (LNB) at room temperature, spin down and mix by pipetting. Due to viscosity, vortexing this buffer is ineffective. Place on ice immediately after thawing and mixing.

Thaw the Elution Buffer (EB) at room temperature and mix by vortexing. Then spin down and place on ice.

Thaw the Short Fragment Buffer (SFB) at room temperature and mix by vortexing. Then spin down and place on ice.

In a 1.5 ml Eppendorf DNA LoBind tube, mix in the following order:

Between each addition, pipette mix 10-20 times.

Reagent Volume cDNA sample from the previous step 60 µl Ligation Buffer (LNB) 25 µl NEBNext Quick T4 DNA Ligase 10 µl Ligation Adapter (LA) 5 µl Total 100 µl

Thoroughly mix the reaction by gently pipetting and briefly spinning down.

Incubate the reaction for 10 minutes at room temperature.

Resuspend the AMPure XP Beads (AXP) by vortexing.