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PCR-cDNA Sequencing Kit

SQK-PCS109

$599.00

Identification and quantification of full-length transcripts with highest throughput

Includes a Flow Cell Priming Kit

Poly-A+ RNA 1 ng 165 mins PCR Throughput
  • 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:

    Feature Property
    Preparation time 165 minutes
    Input requirement 1 ng poly-A+ RNA (or 50 ng total RNA)
    Reverse transcription required Yes
    PCR required Yes
    Read length Enriched for full-length cDNA during PCR
    Read type produced 1D
    Expected data* •••
    Typical number of reads 7–12+ million full-length mapped readsper flow cell on MinION/GridION; 50+ million mapped reads per flow cell on PromethION
    Associated protocols cDNA-PCR Sequencing
    Multiplexing options

    PCR Barcoding Kit

    Flow Cell Wash Kit

    Pack size 6 reactions
    Stability Shipped at  -20° C
    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-ARNA, 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 PolyARNA, 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.

     

  • Protocols
  • Safety and legal
  • What's in the box

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

     

    The Flow Cell Priming Expansion Pack is also supplied.

    EXP-FLP002

  • Multiplexing

    Multiplexing with barcoding for up to 12 samples is available with an additional purchase: PCR Barcoding Kit (SQK-PBK004).

    Alternatively, use the PCR-cDNA Barcoding Kit (SQK-PCB109) instead of the PCR-cDNA Sequencing Kit

  • 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
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:

Feature Property
Preparation time 165 minutes
Input requirement 1 ng poly-A+ RNA (or 50 ng total RNA)
Reverse transcription required Yes
PCR required Yes
Read length Enriched for full-length cDNA during PCR
Read type produced 1D
Expected data* •••
Typical number of reads 7–12+ million full-length mapped readsper flow cell on MinION/GridION; 50+ million mapped reads per flow cell on PromethION
Associated protocols cDNA-PCR Sequencing
Multiplexing options

PCR Barcoding Kit

Flow Cell Wash Kit

Pack size 6 reactions
Stability Shipped at  -20° C
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-ARNA, 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 PolyARNA, 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.

 

What's in the box

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

 

The Flow Cell Priming Expansion Pack is also supplied.

EXP-FLP002

Multiplexing

Multiplexing with barcoding for up to 12 samples is available with an additional purchase: PCR Barcoding Kit (SQK-PBK004).

Alternatively, use the PCR-cDNA Barcoding Kit (SQK-PCB109) instead of the PCR-cDNA Sequencing Kit

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
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