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PCR-cDNA Barcoding Kit
SQK-PCB109

Identification and quantification of full-length transcripts with highest throughput, with barcoding for ≤12 samples

 Includes a Flow Cell Priming Kit
$650.00

Information

This kit is recommended for users who:

  • wish to multiplex samples to reduce price per sample
  • 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 or differential transcript usage
  • want to characterise and quantify isoforms, splice variants and fusion transcripts

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 PCR-cDNA Barcoding Kit features:

FeaturesPCR-cDNA Barcoding Kit
Preparation time165 mins
Input requirement1 ng poly-A+ RNA, or 50 ng total RNA
RT RequiredYes
PCR requiredYes
Read lengthEnriched for full-length cDNA during PCR
Read type produced1D
Typical throughput★★★
Typical number of reads (on FLO-MIN106 at an average read length of 1 kb)7-12+ million full-length mapped reads per flow cell on MinION/GridION; 50+ million mapped reads per flow cell on PromethION
Associated protocolsPCR-cDNA Barcoding (SQK-PCB109)
Pack size6 reactions
StorageShipped 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:

Barcoding or multiplexing is useful when the amount of data required per sample is less than the total amount of data that can be generated from a single flow cell: it allows a user to pool multiple samples and sequence them together, making more efficient use of the flow cell.

The PCR-cDNA Barcoding Kit is used to prepare cDNA for nanopore sequencing for up to 12 samples, 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. The kit contains 12 primer pairs which are used to generate and then amplify double-stranded cDNA by PCR amplification: each primer pair contains a barcode and 5’ tag which facilitates the ligase-free attachment of Rapid Sequencing Adapters. Amplified and barcoded samples are then pooled together and Rapid Sequencing Adapters are added to the pooled mix.

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 poly-A+ RNA, complementary strand synthesis and strand switching are performed using kit-supplied oligonucleotides. The kit contains 12 primer pairs which are used to generate and then amplify double-stranded cDNA by PCR amplification: each primer pair contains a barcode and 5’ tag which facilitates the ligase-free attachment of Rapid Sequencing Adapters. Amplified and barcoded samples are then pooled together, and Rapid Sequencing Adapters are added to the pooled mix. 2019 01 09 SQK PCS109 barcoding workflow v1

Data analysis

Data from a PCR-cDNA sequencing experiment can be analysed using a number of tutorials available in our EPI2ME Labs bioinformatic analysis suite:

  • QC of cDNA reads: A workflow using pychopper providing preliminary analysis and data filtering of Oxford Nanopore Technologies cDNA read datasets
  • Isoform detection: A tutorial guide for identifying full-length transcripts in your cDNA experiments and comparing them against a known annotation
  • Differential gene expression: Pipeline for differential gene expression (DGE) and differential transcript usage (DTU) analysis using nanopore long reads

Protocols

PCR-cDNA Barcoding (SQK-PCB109)

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

What's in the box

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

PCB109 updated

NameAcronymCap colourNo. of vialsFill volume per vial (μl)
VN PrimerVNPBlue1200
Strand Switching PrimersSSPPink1160
Rapid AdapterRAPGreen110
Sequencing BufferSQBRed2300
Sequencing TetherSQTPurple110
Loading BeadsLBPink1360
Elution BufferEBBlack6200
RNA Calibrant StrandRCSYellow125
Barcode Primers 1-12BP01-BP12Clear1210

The Flow Cell Priming Kit is also supplied.

FLP

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

3rd party materials

Consumables

  • Agencourt AMPure XP beads
  • 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
  • 10 mM dNTP solution (e.g. NEB N0447)
  • LongAmp Taq 2X Master Mix (e.g. NEB M0287)
  • Maxima H Minus Reverse Transcriptase (200 U/µl) with 5x RT Buffer (ThermoFisher, cat # EP0751)
  • RNaseOUT™, 40 U/μl (Life Technologies, 10777019)
  • Exonuclease I (NEB, M0293)

Equipment

  • Hula mixer (gentle rotator mixer)
  • Magnetic separator, suitable for 1.5 ml Eppendorf tubes
  • Microfuge
  • Vortex mixer
  • Thermal cycler
  • P1000 pipette and tips
  • P200 pipette and tips
  • P100 pipette and tips
  • P20 pipette and tips
  • P10 pipette and tips
  • P2 pipette and tips
  • Ice bucket with ice
  • Timer
  • Pre-chilled freezer block at -20° C for 200 µl tubes (e.g. Eppendorf 022510509)
  • Qubit fluorometer (or equivalent for QC check)

Compatibility

The PCR-cDNA Barcoding Kit can be used together with:

Kits

  • Flow Cell Wash Kit (EXP-WSH004)

Flow cells

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

Devices

  • MinION Mk1B
  • MinION Mk1C
  • GridION
  • PromethION