- Materials
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- 100 ng PolyA+ RNA, or 70-200 ng already-prepared cDNA
- Direct cDNA Sequencing Kit (SQK-DCS109)
- Flow Cell Priming Kit (EXP-FLP002)
- Consumables
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- Agencourt AMPure XP beads (Beckman Coulter™ cat # A63881)
- NEBNext End repair / dA-tailing Module (E7546)
- NEB Blunt/TA Ligase Master Mix (M0367)
- 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)
- RiboShredder (Epicentre, cat # RS12500), or RNase Cocktail Enzyme Mix (ThermoFisher, cat # AM2286)
- Equipment
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- Hula mixer (gentle rotator mixer)
- Magnetic rack, suitable for 1.5 ml Eppendorf tubes
- Microfuge
- Vortex mixer
- Thermal cycler
- Ice bucket with ice
- Timer
- Pre-chilled freezer block at -20° C for 200 µl tubes (e.g. Eppendorf cat # 022510509)
- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
- P2 pipette and tips
- Optional equipment
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- Qubit fluorometer (or equivalent for QC check)
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For this protocol, you will need 100 ng PolyA+ RNA, or 70-200 ng already-prepared cDNA.
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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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Direct cDNA Sequencing Kit contents
Name Acronym Cap colour No. of vial Fill volume per vial (μl) VN Primer VNP Blue 1 200 Strand Switching Primer SSP Pink 1 160 PR2 Primer PR2 Yellow 1 160 Adapter Mix AMX Green 1 40 Wash Buffer WSB Orange 3 1,000 Elution Buffer EB Black 1 200 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