- Materials
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- Input RNA in 10 mM Tris-HCl, pH 8.0
- Rapid Barcoding Kit 96 (SQK-RBK110.96)
- Consumables
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- LunaScript™ RT SuperMix Kit (NEB, cat # E3010)
- Q5® Hot Start High-Fidelity 2X Master Mix (NEB, cat # M0494)
- SARS-CoV-2 midnight primers (lab-ready at 100 µM, IDT cat # 10007184)
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- Freshly prepared 80% ethanol in nuclease-free water
- Qubit dsDNA HS Assay Kit (ThermoFisher, cat # Q32851)
- DNA 12000 Kit & Reagents - optional (Agilent Technologies)
- 1.5 ml Eppendorf DNA LoBind tubes
- 2 ml Eppendorf DNA LoBind tubes
- 5 ml Eppendorf DNA LoBind tubes
- Eppendorf twin.tec® PCR plate 96 LoBind, semi-skirted (Eppendorf™, cat # 0030129504) with heat seals
- Equipment
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- Hula mixer (gentle rotator mixer)
- Magnetic rack, suitable for 1.5 ml Eppendorf tubes
- Centrifuge capable of taking 96-well plates
- Microfuge
- Vortex mixer
- Thermal cycler
- Multichannel pipettes suitable for dispensing 0.5–10 μl, 2–20 μl and 20–200 μl, and tips
- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
- Ice bucket with ice
- Timer
- Optional equipment
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- Agilent Bioanalyzer (or equivalent)
- Qubit fluorometer (or equivalent for QC check)
- Eppendorf 5424 centrifuge (or equivalent)
- PCR hood with UV steriliser (optional but recommended to reduce cross-contamination)
- PCR-Cooler (Eppendorf)
- Stepper pipette and tips
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For this protocol, you will need your extracted RNA in 16 µl 10 mM Tris-HCl, pH 8.0.
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Rapid Barcoding Kit 96 (SQK-RBK110.96) contents
Name Acronym Cap colour No. of vials Fill volume per vial (µl) Rapid Barcode plate RB96 - 3 plates 8 µl per well AMPure XP Beads AXP Brown 3 1,200 Sequencing Buffer II SBII Red 1 500 Rapid Adapter F RAP-F Green 1 25 Elution Buffer EB Black 1 500 Loading Beads II LBII Pink 1 360 Loading Solution LS White cap, pink label 1 400 Flush Tether FLT Purple 1 400 Flush Buffer FB White 1 bottle 15,500 This product contains AMPure XP reagent manufactured by Beckman Coulter, Inc.
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Rapid barcode sequences
Component Sequence RB01 AAGAAAGTTGTCGGTGTCTTTGTG RB02 TCGATTCCGTTTGTAGTCGTCTGT RB03 GAGTCTTGTGTCCCAGTTACCAGG RB04 TTCGGATTCTATCGTGTTTCCCTA RB05 CTTGTCCAGGGTTTGTGTAACCTT RB06 TTCTCGCAAAGGCAGAAAGTAGTC RB07 GTGTTACCGTGGGAATGAATCCTT RB08 TTCAGGGAACAAACCAAGTTACGT RB09 AACTAGGCACAGCGAGTCTTGGTT RB10 AAGCGTTGAAACCTTTGTCCTCTC RB11 GTTTCATCTATCGGAGGGAATGGA RB12 CAGGTAGAAAGAAGCAGAATCGGA RB13 AGAACGACTTCCATACTCGTGTGA RB14 AACGAGTCTCTTGGGACCCATAGA RB15 AGGTCTACCTCGCTAACACCACTG RB16 CGTCAACTGACAGTGGTTCGTACT RB17 ACCCTCCAGGAAAGTACCTCTGAT RB18 CCAAACCCAACAACCTAGATAGGC RB19 GTTCCTCGTGCAGTGTCAAGAGAT RB20 TTGCGTCCTGTTACGAGAACTCAT RB21 GAGCCTCTCATTGTCCGTTCTCTA RB22 ACCACTGCCATGTATCAAAGTACG RB23 CTTACTACCCAGTGAACCTCCTCG RB24 GCATAGTTCTGCATGATGGGTTAG RB25 GTAAGTTGGGTATGCAACGCAATG RB26 CATACAGCGACTACGCATTCTCAT RB27 CGACGGTTAGATTCACCTCTTACA RB28 TGAAACCTAAGAAGGCACCGTATC RB29 CTAGACACCTTGGGTTGACAGACC RB30 TCAGTGAGGATCTACTTCGACCCA RB31 TGCGTACAGCAATCAGTTACATTG RB32 CCAGTAGAAGTCCGACAACGTCAT RB33 CAGACTTGGTACGGTTGGGTAACT RB34 GGACGAAGAACTCAAGTCAAAGGC RB35 CTACTTACGAAGCTGAGGGACTGC RB36 ATGTCCCAGTTAGAGGAGGAAACA RB37 GCTTGCGATTGATGCTTAGTATCA RB38 ACCACAGGAGGACGATACAGAGAA RB39 CCACAGTGTCAACTAGAGCCTCTC RB40 TAGTTTGGATGACCAAGGATAGCC RB41 GGAGTTCGTCCAGAGAAGTACACG RB42 CTACGTGTAAGGCATACCTGCCAG RB43 CTTTCGTTGTTGACTCGACGGTAG RB44 AGTAGAAAGGGTTCCTTCCCACTC RB45 GATCCAACAGAGATGCCTTCAGTG RB46 GCTGTGTTCCACTTCATTCTCCTG RB47 GTGCAACTTTCCCACAGGTAGTTC RB48 CATCTGGAACGTGGTACACCTGTA RB49 ACTGGTGCAGCTTTGAACATCTAG RB50 ATGGACTTTGGTAACTTCCTGCGT RB51 GTTGAATGAGCCTACTGGGTCCTC RB52 TGAGAGACAAGATTGTTCGTGGAC RB53 AGATTCAGACCGTCTCATGCAAAG RB54 CAAGAGCTTTGACTAAGGAGCATG RB55 TGGAAGATGAGACCCTGATCTACG RB56 TCACTACTCAACAGGTGGCATGAA RB57 GCTAGGTCAATCTCCTTCGGAAGT RB58 CAGGTTACTCCTCCGTGAGTCTGA RB59 TCAATCAAGAAGGGAAAGCAAGGT RB60 CATGTTCAACCAAGGCTTCTATGG RB61 AGAGGGTACTATGTGCCTCAGCAC RB62 CACCCACACTTACTTCAGGACGTA RB63 TTCTGAAGTTCCTGGGTCTTGAAC RB64 GACAGACACCGTTCATCGACTTTC RB65 TTCTCAGTCTTCCTCCAGACAAGG RB66 CCGATCCTTGTGGCTTCTAACTTC RB67 GTTTGTCATACTCGTGTGCTCACC RB68 GAATCTAAGCAAACACGAAGGTGG RB69 TACAGTCCGAGCCTCATGTGATCT RB70 ACCGAGATCCTACGAATGGAGTGT RB71 CCTGGGAGCATCAGGTAGTAACAG RB72 TAGCTGACTGTCTTCCATACCGAC RB73 AAGAAACAGGATGACAGAACCCTC RB74 TACAAGCATCCCAACACTTCCACT RB75 GACCATTGTGATGAACCCTGTTGT RB76 ATGCTTGTTACATCAACCCTGGAC RB77 CGACCTGTTTCTCAGGGATACAAC RB78 AACAACCGAACCTTTGAATCAGAA RB79 TCTCGGAGATAGTTCTCACTGCTG RB80 CGGATGAACATAGGATAGCGATTC RB81 CCTCATCTTGTGAAGTTGTTTCGG RB82 ACGGTATGTCGAGTTCCAGGACTA RB83 TGGCTTGATCTAGGTAAGGTCGAA RB84 GTAGTGGACCTAGAACCTGTGCCA RB85 AACGGAGGAGTTAGTTGGATGATC RB86 AGGTGATCCCAACAAGCGTAAGTA RB87 TACATGCTCCTGTTGTTAGGGAGG RB88 TCTTCTACTACCGATCCGAAGCAG RB89 ACAGCATCAATGTTTGGCTAGTTG RB90 GATGTAGAGGGTACGGTTTGAGGC RB91 GGCTCCATAGGAACTCACGCTACT RB92 TTGTGAGTGGAAAGATACAGGACC RB93 AGTTTCCATCACTTCAGACTTGGG RB94 GATTGTCCTCAAACTGCCACCTAC RB95 CCTGTCTGGAAGAAGAATGGACTT RB96 CTGAACGGTCATAGAGTCCACCAT