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:

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.

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.

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

Safety Data Sheet for the PCR-cDNA Sequencing Kit (SQK-PCS109)

Safety Data Sheet for Flow Cell Priming Kit

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

The Flow Cell Priming Expansion Pack is also supplied.

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

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)

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
• GridION X5
• PromethION