- Materials
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- Barcode Primers (BP01-24)
- Elution Buffer (EB)
- Consumables
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- Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
- LongAmp Hot Start Taq 2X Master Mix (NEB, M0533S)
- Exonuclease I (NEB, Cat # M0293)
- Agencourt AMPure XP beads (Beckman Coulter™ cat # A63881)
- Freshly prepared 70% ethanol in nuclease-free water
- 0.2 ml PCR tubes
- 1.5 ml Eppendorf DNA LoBind tubes
- Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- Equipment
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- Thermal cycler
- Vortex mixer
- Hula mixer (gentle rotator mixer)
- Magnetic rack, suitable for 1.5 ml Eppendorf tubes
- 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
- Qubit fluorometer (or equivalent for QC check)
- Agilent Bioanalyzer (or equivalent)
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Thaw the following reagents, then spin down briefly using a microfuge and mix as indicated in the table below. Then place the reagents on ice.
Reagent 1. Thaw at room temperature 2. Briefly spin down 3. Mix well by pipetting Barcode Primers (BP01 - BP24) ✓ ✓ ✓ Elution Buffer (EB) ✓ ✓ ✓ LongAmp Hot Start Taq 2X Master Mix ✓ ✓ ✓ Exonuclease I Not frozen ✓ ✓ -
Spin down the reverse-transcribed RNA samples.
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Prepare a separate 0.2 ml PCR tube for each sample and add 5 μl of reverse-transcribed RNA per tube.
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In each of the 0.2 ml PCR tubes containing reverse-transcribed RNA sample, prepare the following reaction at room temperature:
Reagent Volume Reverse-transcribed sample (from previous step) 5 μl Unique Barcode Primer (BP01-24) 0.75 μl Nuclease-free water 6.75 μl 2x LongAmp Hot Start Taq Master Mix 12.5 μl Total (including all reagents) 25 μl -
Mix gently by pipetting.
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Amplify using the following cycling conditions.
Cycle step Temperature Time No. of cycles Initial denaturation 95°C 30 secs 1 Denaturation 95°C 15 secs 10-18* Annealing 62°C 15 secs 10-18* Extension 65°C 60 secs per kb 10-18* Final extension 65°C 6 mins 1 Hold 4°C ∞ *We recommend 14 cycles as a starting point. However, the number of cycles can be adjusted between the values shown according to experimental needs.
For further information, please read The effect of varying the number of PCR cycles in the PCR-cDNA Sequencing Kit document.
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Add 1 μl Exonuclease I directly to each PCR tube. Mix by pipetting.
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Incubate the reactions at 37°C for 15 minutes, followed by 80°C for 15 minutes in the thermal cycler.
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Transfer each sample to a clean 1.5 ml Eppendorf DNA LoBind tube.
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Resuspend the AMPure XP beads by vortexing.
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Add 20 µl of resuspended AMPure XP beads to each 1.5 ml Eppendorf DNA LoBind tube.
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Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.
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Prepare 5 ml of fresh 70% ethanol in nuclease-free water.
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Spin down the samples and pellet on a magnet. Keep the tubes on the magnet, and pipette off the supernatant.
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Keep the tubes on the magnet and wash the beads with 200 µl of freshly-prepared 70% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
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Repeat the previous step.
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Spin down and place the tubes back on the magnet. Pipette off any residual ethanol. Allow to dry for ~30 seconds, but do not dry the pellets to the point of cracking.
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Remove the tubes from the magnetic rack and resuspend each pellet in 12 µl of Elution Buffer (EB).
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Incubate at room temperature for 10 minutes.
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Pellet the beads on the magnet until the eluate is clear and colourless.
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Remove and retain 12 µl of each eluate into a separate clean 1.5 ml Eppendorf DNA LoBind tube.
- Remove and retain the eluate which contains the cDNA library in a clean 1.5 ml Eppendorf DNA LoBind tube
- Dispose of the pelleted beads
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For each sample, analyse 1 µl of the amplified cDNA for size, quantity and quality using a Qubit fluorometer and Agilent Bioanalyzer (or equivalent) for a QC check.
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Pool together equimolar samples of the amplified cDNA barcoded samples to a total of 15 – 25 fmols and make the volume up to 23 µl in Elution Buffer (EB).
Mass Molarity if fragment length = 0.5 kb Molarity if fragment length = 1.5 kb Molarity if fragment length = 3 kb 5 ng 16 fmol 5 fmol 3 fmol 10 ng 32 fmol 11 fmol 5 fmol 15 ng 49 fmol 16 fmol 8 fmol 20 ng 65 fmol 22 fmol 11 fmol 25 ng 81 fmol 27 fmol 13 fmol 50 ng 154 fmol 51 fmol 26 fmol If the quantity of amplified cDNA is above 25 fmol, the remaining cDNA can be frozen and stored for another sequencing experiment (in this case, library preparation would start from the Adapter Addition step). We recommend avoiding multiple freeze-thaw cycles to prevent DNA degradation.
The sequencing adapter used in Kit 11 chemistry has a higher capture rate, enabling lower flow cell loading amounts to give optimal pore occupancy.