- Materials
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- 4 ng enriched RNA (Poly(A)+ RNA or ribodepleted) or 200 ng total RNA
- PCR-cDNA Barcoding Kit (SQK-PCB111.24)
- Consumables
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- Agencourt RNAClean XP beads (Beckman Coulter™, cat # A63987)
- Agencourt AMPure XP beads (Beckman Coulter™ cat # A63881)
- Lambda Exonuclease (NEB, Cat # M0262L)
- NEBNext® Quick Ligation Reaction Buffer (NEB, B6058)
- T4 DNA Ligase 2M U/ml (NEB, cat # M0202M)
- 10 mM dNTP solution (e.g. NEB N0447)
- LongAmp Hot Start Taq 2X Master Mix (NEB, M0533S)
- Maxima H Minus Reverse Transcriptase (200 U/µl) with 5x RT Buffer (ThermoFisher, cat # EP0751)
- RNaseOUT™, 40 U/μl (Life Technologies, cat # 10777019)
- USER (Uracil-Specific Excision Reagent) Enzyme (NEB, cat # M5505L)
- Exonuclease I (NEB, Cat # M0293)
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- Freshly prepared 70% ethanol in nuclease-free water
- 1.5 ml Eppendorf DNA LoBind tubes
- 0.2 ml thin-walled PCR tubes
- Qubit RNA HS Assay Kit (ThermoFisher, cat # Q32852)
- Qubit dsDNA HS Assay Kit (ThermoFisher, cat # Q32851)
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- Equipment
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- 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 4 ng enriched RNA (Poly(A)+ RNA or ribodepleted) or 200 ng total RNA.
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Input RNA
It is important that the input RNA meets the quantity and quality requirements. Using too little or too much RNA, or RNA of poor quality (e.g. fragmented or containing chemical contaminants) can affect your library preparation.
For instructions on how to perform quality control of your RNA sample, please read the Input DNA/RNA QC protocol.
For further information on using RNA as input, please read the links below.
- Polyadenylation of non-poly(A) transcripts using E. coli poly(A) polymerase
- RNA contaminants
- RNA stability
- RNA Integrity Number (RIN)
- Enrichment of polyadenylated RNA molecules
These documents can also be found in the DNA/RNA Handling page.
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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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PCR-cDNA Barcoding Kit (SQK-PCB111.24) 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 T RAP T Green 1 10 RAP Dilution Buffer
(or Adapter Buffer)RDB
(or ADB)Clear 1 100 Elution Buffer EB Black 2 1,500 Short Fragment Buffer SFB White 4 7,500 Sequencing Buffer II SBII Red 1 500 Loading Beads II LBII Pink 1 360 Loading Solution LS White cap, pink label 1 400 Barcode Primers 1-24 BP01-24 White 24 10 Flush Buffer FB Blue 6 1,170 Flush Tether FLT White cap, purple label 1 200 -
RNA Control Expansion (EXP-RCS001)
The RNA Control Expansion (EXP-RCS001) provides users with RNA Control Sample (RCS) to replace their sample input when performing a control experiment for troubleshooting purposes in the cDNA-PCR Sequencing (SQK-PCS111) and the PCR-cDNA Barcoding Kit (SQK-PCB111.24).
Name Acronym Number of vials Cap colour Fill volume per vial (µl) RNA Control Sample RCS 3 Yellow 25 -
Barcode primer sequences
Below are the barcode primer sequences for PCR-cDNA Barcoding Kit (SQK-PCB109 and SQK-PCB111.24).
Component Forward sequence Reverse sequence BP01 CAAGAAAGTTGTCGGTGTCTTTGTGAC CAAGAAAGTTGTCGGTGTCTTTGTGTT BP02 CTCGATTCCGTTTGTAGTCGTCTGTAC CTCGATTCCGTTTGTAGTCGTCTGTTT BP03 CGAGTCTTGTGTCCCAGTTACCAGGAC CGAGTCTTGTGTCCCAGTTACCAGGTT BP04 CTTCGGATTCTATCGTGTTTCCCTAAC CTTCGGATTCTATCGTGTTTCCCTATT BP05 CCTTGTCCAGGGTTTGTGTAACCTTAC CCTTGTCCAGGGTTTGTGTAACCTTTT BP06 CTTCTCGCAAAGGCAGAAAGTAGTCAC CTTCTCGCAAAGGCAGAAAGTAGTCTT BP07 CGTGTTACCGTGGGAATGAATCCTTAC CGTGTTACCGTGGGAATGAATCCTTTT BP08 CTTCAGGGAACAAACCAAGTTACGTAC CTTCAGGGAACAAACCAAGTTACGTTT BP09 CAACTAGGCACAGCGAGTCTTGGTTAC CAACTAGGCACAGCGAGTCTTGGTTTT BP10 CAAGCGTTGAAACCTTTGTCCTCTCAC CAAGCGTTGAAACCTTTGTCCTCTCTT BP11 CGTTTCATCTATCGGAGGGAATGGAAC CGTTTCATCTATCGGAGGGAATGGATT BP12 CCAGGTAGAAAGAAGCAGAATCGGAAC CCAGGTAGAAAGAAGCAGAATCGGATT BP13 CAGAACGACTTCCATACTCGTGTGAAC CAGAACGACTTCCATACTCGTGTGATT BP14 CAACGAGTCTCTTGGGACCCATAGAAC CAACGAGTCTCTTGGGACCCATAGATT BP15 CAGGTCTACCTCGCTAACACCACTGAC CAGGTCTACCTCGCTAACACCACTGTT BP16 CCGTCAACTGACAGTGGTTCGTACTAC CCGTCAACTGACAGTGGTTCGTACTTT BP17 CACCCTCCAGGAAAGTACCTCTGATAC CACCCTCCAGGAAAGTACCTCTGATTT BP18 CCCAAACCCAACAACCTAGATAGGCAC CCCAAACCCAACAACCTAGATAGGCTT BP19 CGTTCCTCGTGCAGTGTCAAGAGATAC CGTTCCTCGTGCAGTGTCAAGAGATTT BP20 CTTGCGTCCTGTTACGAGAACTCATAC CTTGCGTCCTGTTACGAGAACTCATTT BP21 CGAGCCTCTCATTGTCCGTTCTCTAAC CGAGCCTCTCATTGTCCGTTCTCTATT BP22 CACCACTGCCATGTATCAAAGTACGAC CACCACTGCCATGTATCAAAGTACGTT BP23 CCTTACTACCCAGTGAACCTCCTCGAC CCTTACTACCCAGTGAACCTCCTCGTT BP24 CGCATAGTTCTGCATGATGGGTTAGAC CGCATAGTTCTGCATGATGGGTTAGTT