- Materials
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- 300 ng of poly(A) tailed RNA or 1 µg of total RNA in 8 µl
- RT Adapter (RTA)
- RNA CS (RCS)
- Wash Buffer (WSB)
- RNA Ligation Adapter (RLA)
- RNA Elution Buffer (REB)
- Consumables
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- Agencourt RNAClean XP beads (Beckman Coulter™, cat # A63987)
- SuperScript III Reverse Transcriptase, 5x First-strand buffer and 100 mM DTT (Thermo Fisher Scientific, cat # 18080044)
- 10 mM dNTP solution (e.g. NEB, cat # N0447)
- NEBNext® Quick Ligation Reaction Buffer (NEB, B6058)
- T4 DNA Ligase 2M U/ml (NEB, cat # M0202T/M)
- RNaseOUT™ Recombinant Ribonuclease Inhibitor (Invitrogen, 10777019)
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- Freshly prepared 70% ethanol in nuclease-free water
- 0.2 ml thin-walled PCR tubes
- 1.5 ml Eppendorf DNA LoBind tubes
- Qubit 1x dsDNA HS Assay Kit (ThermoFisher, Q33230)
- Qubit RNA HS Assay Kit (ThermoFisher, cat # Q32852)
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- Equipment
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- Hula mixer (gentle rotator mixer)
- Thermal cycler
- Magnetic rack
- Qubit fluorometer (or equivalent for QC check)
- Ice bucket with ice
- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
- P2 pipette and tips
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Prepare the NEBNext Quick Ligation Reaction Buffer and T4 DNA Ligase according to the manufacturer's instructions, and place on ice:
Thaw the reagents at room temperature.
Spin down the reagent tubes for 5 seconds.
Ensure the reagents are fully mixed by performing 10 full volume pipette mixes.
Note: Do NOT vortex the T4 DNA Ligase.
The NEBNext Quick Ligation Reaction 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 several seconds to ensure the reagent is thoroughly mixed.
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Spin down the RT Adapter (RTA), RNA CS (RCS) (if using), and RNA Ligation Adapter (RLA), pipette mix and place on ice.
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Thaw the Wash Buffer (WSB) and RNA Elution Buffer (REB) at room temperature and mix by vortexing. Then spin down and place on ice.
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Prepare the RNA in nuclease-free water:
- Transfer 300 ng of poly(A) tailed RNA or 1 µg of total RNA into a 0.2 ml thin-walled PCR tube.
- Adjust the volume to 8 μl with nuclease-free water.
- Mix thoroughly by flicking the tube to avoid unwanted shearing.
- Spin down briefly in a microfuge.
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Optional actionThe use of the RNA CS (RCS) in this preparation is an optional control measure for library preparation QC and troubleshooting.
We recommend the inclusion of the RNA CS (RCS) in the library preparation for troubleshooting purposes.
However, if users chose to not use the RNA CS (RCS) input, replace the volume in the reaction with nuclease-free water.
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In the same 0.2 ml thin-walled PCR tube, combine the reagents in the following order:
Reagent Volume RNA 8 µl NEBNext Quick Ligation Reaction Buffer 3 µl RNA CS (RCS) 0.5 µl RNaseOUT™ 1 µl RT Adapter (RTA) 1 µl T4 DNA Ligase 1.5 µl Total 15 µl -
Mix by pipetting and spin down.
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Incubate the reaction for 10 minutes at room temperature.
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In a clean 1.5 ml DNA LoBind Eppendorf tube, combine the following reagents together to make the reverse transcription master mix:
Reagent Volume Nuclease-free water 9 µl 10 mM dNTPs 2 µl 5X First-strand buffer 8 µl DTT 4 µl Total 23 µl -
Transfer the reverse transcriptase master mix to the 0.2 ml PCR tube containing your adapter-ligated RNA and mix by pipetting.
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Add 2 µl of SuperScript III Reverse Transcriptase to the reaction and mix by pipetting.
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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.
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Transfer the sample to a clean 1.5 ml Eppendorf DNA LoBind tube.
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Resuspend the stock of Agencourt RNAClean XP beads by vortexing.
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Add 72 µl of resuspended Agencourt RNAClean XP beads to the reverse transcription reaction and mix by pipetting.
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Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.
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Prepare 200 μl of fresh 70% ethanol in nuclease-free water.
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Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant when clear and colourless.
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Keep the tube on magnet until the supernatant is clear and colourless before washing the beads with 150 µl of freshly prepared 70% ethanol, as described below:
- Keeping the magnetic rack on the benchtop, rotate the tube by 180°. Wait for the beads to migrate towards the magnet and to form a pellet.
- Rotate the tube 180° again (back to the starting position), and wait for the beads to pellet again.
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Carefully remove the 70% ethanol using a pipette and discard.
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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.
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Remove the tube from the magnetic rack and resuspend the pellet in 23 µl nuclease-free water. Incubate for 5 minutes at room temperature.
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Pellet the beads on a magnet until the eluate is clear and colourless.
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Remove and retain 23 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
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Optional actionAt this stage the RT-RNA sample can be stored at -80°C for later use.
Please note, this is the only pause point in this protocol.
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In the same 1.5 ml Eppendorf DNA LoBind tube, combine the reagents in the following order:
Reagent Volume RT-RNA sample 23 µl NEBNext Quick Ligation Reaction Buffer 8 µl RNA Ligation Adapter (RLA) 6 µl T4 DNA Ligase 3 µl Total 40 µl -
Mix by pipetting.
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Incubate the reaction for 10 minutes at room temperature.
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Resuspend the stock of Agencourt RNAClean XP beads by vortexing.
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Add 16 µl of resuspended Agencourt RNAClean XP beads to the reaction and mix by pipetting.
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Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.
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Spin down the sample and pellet on a magnet. Keep the tube on the magnet for 5 minutes, and pipette off the supernatant when clear and colourless.
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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 for 5 minutes and pipette off the supernatant when clear and colourless.
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Repeat the previous step.
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Spin down the tube and replace onto the magnetic rack until the beads have pelleted to pipette off any remaining Wash Buffer (WSB).
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Remove the tube from the magnetic rack and resuspend the pellet in 13 µl RNA Elution Buffer (REB) by the gently flicking the tube. Incubate for 10 minutes at room temperature.
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Pellet the beads on a magnet for 5 minutes until the eluate is clear and colourless.
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Remove and retain 13 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
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Quantify 1 µl of reverse-transcribed and adapted RNA using the Qubit fluorometer DNA HS assay.
The recovery aim in the final eluate is > 30 ng.
Recovery quantities can vary between different inputs and library preparations. However, we always recommend taking forward the full volume of RNA library for the best sequencing results.