- Materials
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- 10 ng enriched RNA (Poly(A)+ RNA or ribodepleted) or 500 ng total RNA per sample
- cDNA-PCR Barcoding Kit 24 V14 (SQK-PCB114.24)
- Consumables
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- MinION and GridION Flow Cell
- NEBNext® Quick Ligation Reaction Buffer (NEB, B6058)
- T4 DNA Ligase 2M U/ml (NEB, M0202M)
- RNaseOUT™, 40 U/μl (Life Technologies, cat # 10777019)
- Lambda Exonuclease (NEB, Cat # M0262L)
- Thermolabile Exonuclease I (NEB, cat # M0568)
- USER (Uracil-Specific Excision Reagent) Enzyme (NEB, cat # M5505L)
- 10 mM dNTP solution (e.g. NEB N0447)
- Maxima H Minus Reverse Transcriptase (200 U/µl) with 5x RT Buffer (ThermoFisher, cat # EP0752)
- LongAmp Hot Start Taq 2X Master Mix (NEB, M0533S)
- Agencourt RNAClean XP beads (Beckman Coulter™, cat # A63987)
- Agencourt AMPure XP beads (Beckman Coulter™ cat # A63881)
- Bovine Serum Albumin (BSA) (50 mg/ml) (e.g Invitrogen™ UltraPure™ BSA 50 mg/ml, AM2616)
- Qubit dsDNA HS Assay Kit (ThermoFisher, cat # Q32851)
- Qubit RNA HS Assay Kit (ThermoFisher, cat # Q32852)
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- Freshly prepared 70% ethanol in nuclease-free water
- 1.5 ml Eppendorf DNA LoBind tubes
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- 0.2 ml thin-walled PCR tubes
- Equipment
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- MinION or GridION device
- MinION and GridION Flow Cell Light Shield
- Hula mixer (gentle rotator mixer)
- Magnetic rack, suitable for 1.5 ml Eppendorf tubes
- Microfuge
- Vortex mixer
- Thermal cycler
- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
- P2 pipette and tips
- Ice bucket with ice
- Timer
- Qubit fluorometer (or equivalent for QC check)
- Agilent Bioanalyzer (or equivalent)
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For this protocol, you will need 10 ng enriched RNA (Poly(A)+ RNA or ribodepleted) or 500 ng total RNA per sample.
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Third-party reagents
We have validated and recommend the use of all the third-party reagents used in this protocol. Alternatives have not been tested by Oxford Nanopore Technologies.
For all third-party reagents, we recommend following the manufacturer's instructions to prepare the reagents for use.
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Check your flow cell
We highly recommend that you check the number of pores in your flow cell prior to starting a sequencing experiment. This should be done within three months of purchasing for MinION/GridION/PromethION or within four weeks of purchasing Flongle Flow Cells. Oxford Nanopore Technologies will replace any flow cell with fewer than the number of pores in the table below, when the result is reported within two days of performing the flow cell check, and when the storage recommendations have been followed. To do the flow cell check, please follow the instructions in the Flow Cell Check document.
Flow cell Minimum number of active pores covered by warranty Flongle Flow Cell 50 MinION/GridION Flow Cell 800 PromethION Flow Cell 5000 -
cDNA-PCR Barcoding Kit 24 V14 (SQK-PCB114.24) contents
Name Acronym Cap colour No. of vials Fill volume per vial (μl) Strand Switching Primer II SSPII Violet 1 350 RT Primer RTP Yellow 1 200 cDNA RT Adapter CRTA Amber 1 200 Annealing Buffer AB Orange 1 200 Rapid Adapter RA Green 1 15 Adapter Buffer ADB Clear 1 100 Elution Buffer EB Black 2 500 Short Fragment Buffer SFB Clear 4 7,500 Sequencing Buffer SB Red 1 700 Library Beads LIB Pink 1 600 Library Solution LIS White cap, pink label 1 600 Barcode Primers 1-24 BP01-24 White 24 10 Flow Cell Tether FCT Purple 1 200 Flow Cell Flush FCF Clear cap, light blue label 1 8,000