- Materials
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- 1 ng PolyA+ RNA (or ~50 ng total RNA)
- cDNA-PCR Sequencing Kit (SQK-PCS109)
- Flow Cell Priming Kit (EXP-FLP002)
- Consumables
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- Agencourt AMPure XP beads (Beckman Coulter™ cat # A63881)
- 1.5 ml Eppendorf DNA LoBind tubes
- 0.2 ml thin-walled PCR tubes
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- Freshly prepared 70% ethanol in nuclease-free water
- 10 mM dNTP solution (e.g. NEB N0447)
- LongAmp Taq 2X Master Mix (e.g. NEB M0287)
- Maxima H Minus Reverse Transcriptase (200 U/µl) with 5x RT Buffer (ThermoFisher, cat # EP0751)
- RNaseOUT™, 40 U/μl (Life Technologies, cat # 10777019)
- Exonuclease I (NEB, Cat # M0293)
- 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
- Pre-chilled freezer block at -20° C for 200 µl tubes (e.g. Eppendorf cat # 022510509)
- Qubit fluorometer (or equivalent for QC check)
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For this protocol, you will need 1 ng PolyA+ RNA, or ~50 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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cDNA-PCR Sequencing Kit contents
Name Acronym Cap colour No. of vials Fill volume per vial (μl) VN Primer VNP Blue 1 200 Strand Switching Primer SSP Pink 1 160 cDNA Primer cPRM Grey 1 40 Elution Buffer EB Black 2 200 Rapid Adapter RAP Green 1 10 Sequencing Buffer SQB Red 2 300 Loading Beads LB Pink 1 360 -
Flow Cell Priming Kit contents (EXP-FLP002)
Name Acronym Cap colour No. of vials Fill volume per vial (μl) Flush Buffer FB Blue 6 1,170 Flush Tether FLT Purple 1 200