Direct cDNA Sequencing Kit
SQK-DCS109

Identification and quantification of full-length transcripts without PCR bias

 Includes a Flow Cell Priming Kit

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

The Direct cDNA Sequencing Kit features:

FeatureProperty
Preparation time270 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
Expected data*•••
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 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 -20°C, Long-term storage -20°CCell

Description

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.

Further considerations:

A Native Barcoding Expansion pack (EXP-NBD104) can be used to multiplex up to 12 different RNA samples into a single flow cell.

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

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

What's in the box

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

2019 01 25 SQK-DCS109 contents v2

The Flow Cell Priming Expansion Pack is also supplied.

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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-WSH003)

Flow cells

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

Devices

  • MinION Mk 1B
  • MinION Mk 1C
  • GridION Mk1
  • PromethION