PCR-cDNA Sequencing Kit
SQK-PCS109

Identification and quantification of full-length transcripts with highest throughput

 Includes a Flow Cell Priming Kit

Information

This kit is highly recommended for users who:

  • have a limited amount of input material
  • want to optimise their sequencing experiment for throughput
  • 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

The PCR-cDNA Sequencing Kit features:

FeatureProperty
Preparation time165 minutes
165 minutes1 ng poly-A+ RNA (or 50 ng total RNA)
Reverse transcription requiredYes
PCR requiredYes
Read lengthEnriched for full-length cDNA during PCR
Read type produced1D
Expected data*•••
Typical number of reads7–12+ million full-length mapped readsper flow cell on MinION/GridION; 50+ million mapped reads per flow cell on PromethION
Associated protocolscDNA-PCR Sequencing
Multiplexing optionsPCR Barcoding Kit and Flow Cell Wash Kit
Pack size6 reactions
StabilityShipped at -20° C and Long-term storage -20° C

Description

The PCR-cDNA Sequencing Kit is used to prepare cDNA for nanopore sequencing from an input of as low as 1 ng poly-A+ RNA. Users who do not have poly-A+ enriched RNA can use 50 ng of total RNA but additional optimisation may be required.

Taking full-length poly-A+ RNA, complementary strand synthesis and strand switching are performed using kit-supplied oligonucleotides. dscDNA is then generated by PCR amplification using primers that contain 5’ tags which facilitate the ligase-free attachment of Rapid Sequencing Adapters.

Further considerations

The PCR Barcoding Kit (SQK-PBK004) 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 PolyA+ RNA, complementary strand synthesis and strand switching are performed using kit-supplied oligonucleotides. dscDNA is then generated by PCR amplification using primers that contain 5’ tags which facilitate the ligase-free attachment of Rapid Sequencing Adapters.

2019 01 07 SQK PCS109 workflow v1

Protocols

Example protocol using the PCR-cDNA Sequencing Kit (SQK-PCS109) - for illustration purposes only

Example protocol using the PCR-cDNA Sequencing Kit (SQK-PCS109) - PromethION - for illustration purposes only

What's in the box

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

2019 01 25 SQK-PCS109 contents v2

The Flow Cell Priming Expansion Pack is also supplied.

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

3rd party materials

Consumables

  • 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)
  • Exonuclease I (NEB, cat # M0293)

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 PCR-cDNA Sequencing Kit can be used together with:

Kits

  • PCR Barcoding Kit (SQK-PBK004)
  • Flow Cell Wash Kit (EXP-WSH003)

Flow cells

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

Devices

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