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Nanopore sequencing offers advantages in all areas of research. Our offering includes DNA sequencing, as well as RNA and gene expression analysis and future technology for analysing proteins.

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Direct cDNA Sequencing Kit
SQK-DCS109

Identification and quantification of full-length transcripts without PCR bias

 Includes a Flow Cell Priming Kit ll
£485.00

Information

This kit is highly recommended for users who:

  • would like to identify and quantify full-length transcripts
  • are interested in differential gene expression
  • want to characterise and quantify isoforms, splice variants and fusion transcripts
  • wish to avoid bias and artefacts introduced through PCR

Please note that to use this kit, you will need to purchase additional 3rd party reagents: see the "3rd Party Materials" tab for more detail.

The Direct cDNA Sequencing Kit is used to prepare cDNA for nanopore sequencing without using PCR. The kit recommends an input of 100 ng poly-A+RNA.

Taking full-length poly-A+ RNA, complementary strand synthesis and strand switching are performed using kit-supplied oligonucleotides. The RNA template is then digested, and the second (complementary) strand is synthesised. Sequencing adapters supplied in the Direct cDNA Sequencing Kit are then ligated onto the dscDNA.

The Direct cDNA Sequencing Kit features:

FeatureProperty
Preparation time275 minutes
Input requirement100 ng poly-A+ RNA
Reverse transcription requiredYes
PCR requiredNo
Read lengthEnriched for ful-length cDNA during second strand synthesis
Read type produced1D
Typical throughput★★☆
Typical number of reads5-10 million full-length per flow cell on MinION/GirdION; 15-30 million per flow cell on PromethION
Associated protocolsDirect cDNA sequencing
Direct cDNA native barcoding
Multiplexing options• Native Barcoding Expansion Pack 1-12 (EXP-NBD104)
• Native Barcoding Expansion Pack 13-24 (EXP-NBD114)
• Flow Cell Wash Kit
Pack size6 reactions
StabilityShipped at 2–8°C

Long-term storage -20°C

Oxford Nanopore Technologies deem the useful life of the product to be 3 months from receipt by the customer.

Further considerations:

The Native Barcoding Expansion packs 1-12 (EXP-NBD104) and 13-24 (EXP-NBD114) can be used to multiplex up to 24 different RNA samples into a single flow cell.

Shipping and logistics:

Flow cells and kits are shipped together at 2–8°C.

Upon receipt, please place the right product in the right long-term storage location.

Products are shipped to customers within the USA and EU Monday to Thursday. Shipments to Canada, Norway, Korea and Japan are expedited Monday to Wednesday; with Australia and New Zealand leaving our warehouses on a Friday. Shipments to the rest of the world are made on Mondays to allow the full working week for packages to arrive.

The delivery charges are calculated when a quote is raised or during checkout. Once an order is made, the delivery ID and delivery information can be tracked in the Store.

Workflow

Taking full-length messenger RNA, the complementary strand is synthesised using a kit-supplied oligo adapter. The RNA is then degraded, and the second (complementary) strand synthesised. Adapters supplied in the Direct cDNA Sequencing Kit are then ligated onto the cDNA, which introduce the components needed for the cDNA strands to enter the pore. One strand of the duplex is sequenced at a time, producing 1D reads.

2019 01 07 SQK DCS109 workflow v1

Protocols

Direct cDNA sequencing
Direct cDNA native barcoding

To access the protocols, you will need to register for a Nanopore Community account.

What's in the box

The Direct cDNA Sequencing Kit contains sufficient reagents to generate six sequencing libraries.

2021 01 05 SQK-DCS109 contents v3

NameAcronymCap colourNo. of vialFill volume per vial (μl)
VN PrimerVNPBlue1200
Strand Switching PrimerSSPPink1160
PR2 PrimerPR2Yellow1160
Adapter MixAMXGreen140
Wash BufferWSBOrange31,000
Elution BufferEBBlack1200
Sequencing BufferSQBRed2300
Sequencing TetherSQTPurple110
Loading BeadsLBPink1360

The Flow Cell Priming Kit is also supplied.

FLPEXP-FLP002({{media url= "EXP-FLP002")

NameAcronymCap colourNo. of vialsFill volume per vial (μl)
Flush BufferFBBlue61,170
Flush TetherFLTPurple1200

3rd party materials

Consumables

  • 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, cat # AM9937)
  • Freshly prepared 70% ethanol in nuclease-free water
  • Agencourt AMPure XP beads
  • 10 mM dNTP solution (e.g. NEB cat # N0447)
  • LongAmp Taq 2X Master Mix (e.g. NEB cat # 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, RS12500), or RNase Cocktail Enzyme Mix (ThermoFisher, cat # AM2286)

Equipment

  • Hula mixer (gentle rotator mixer)
  • Magnetic rack
  • Microfuge
  • Vortex mixer
  • Ice bucket with ice
  • Timer
  • Thermal cycler
  • P1000 pipette and tips
  • P100 pipette and tips
  • P20 pipette and tips
  • P10 pipette and tips
  • P2 pipette and tips
  • Pre-chilled freezer block at -20°C for 200 µl tubes (e.g. Eppendorf cat # 022510509)

Optional equipment

  • Agilent Bioanalyzer (or equivalent)
  • Qubit fluorometer (or equivalent for QC check)
  • Eppendorf 5424 centrifuge (or equivalent)

Compatibility

The Direct cDNA Sequencing Kit can be used together with:

Kits

  • Native Barcoding Expansion 1-12 (EXP-NBD104)
  • Native Barcoding Expansion 13-24 (EXP-NBD114)
  • Flow Cell Wash Kit (EXP-WSH004)

Flow cells

  • FLO-MINSP6
  • FLO-MIN106D
  • FLO-PRO002

Devices

  • MinION Mk1B
  • MinION Mk1C
  • GridION
  • PromethION