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Nanopore sequencing offers advantages in all areas of research. Our offering includes DNA sequencing, as well as RNA and gene expression analysis and future technology for analysing proteins.

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Oxford Nanopore TechnologiesFully scalable, real-time DNA/RNA sequencing technology
Oxford Nanopore DiagnosticsLamPORE – rapid, low-cost, scalable detection of SARS-CoV-2
London Calling 2022
18th - 20th May
cDNA-PCR Sequencing Kit
SQK-PCS111

Identification and quantification of full-length transcripts with highest output.

This product has a 6 week lead time

$599.00

Information

This kit is recommended for users who:

  • have a limited amount of input material
  • want to optimise their sequencing experiment for output
  • 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 cDNA-PCR Sequencing Kit features:

FeaturesProperties
Preparation time~160 mins + PCR
Input requirement4 ng poly-A+ RNA, or 200 ng total RNA
RT RequiredYes
PCR requiredYes
Read lengthEnriched for full-length cDNA during PCR
Read type produced1D
Associated protocolsPCR-cDNA Sequencing Kit (SQK-PCS111)
Multiplexing optionsFlow Cell Wash Kit (EXP-WSH004)
Pack size6 reactions

The PCR-cDNA Sequencing Kit is used to prepare cDNA for nanopore sequencing from an input of as low as 4 ng poly-A+ RNA. Users who do not have poly-A+ enriched RNA can use 200 ng of total RNA but additional optimisation may be required.

The protocol uses a strand switching method to select for full length transcripts, allowing the identification of splice variants. Taking full-length poly-A+ RNA, complementary strand synthesis and strand switching are performed using kit-supplied oligonucleotides. Double-stranded cDNA is then generated by PCR amplification using primers that contain 5’ tags which facilitate the ligase-free attachment of Rapid Sequencing Adapters.

This cDNA kit (Kit 11) includes the Rapid Adapter T (RAP T), with two key features: Firstly, the adapter is higher capture, enabling lower flow cell loading amounts. Secondly, the adapter contains the fuel fix technology, enabling users to run long experiments without the need for fuel addition during the run.

The PCR-cDNA Sequencing Kit also includes a new cDNA RT adapter and RT primer to prime cDNA synthesis from the end of a transcript to reduce overlaps during the reverse transcription step and to allow users to measure polyA+ tail lengths.

Shipping and logistics:

Flow cells and kits are shipped together in an environmentally friendly temperature-controlled shipping box.

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. Double-stranded cDNA is then generated by PCR amplification using primers that contain 5’ tags which facilitate the ligase-free attachment of Rapid Sequencing Adapters.

PCS111 workflow

Protocols

PCR-cDNA Sequencing Kit (SQK-PCS111)

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

What's in the box

The cDNA-PCR Sequencing Kit (SQK-PCS111) kit contains enough reagents for six reactions:

SQK-PCS111 tubes

NameAcronymCap colourNo. of vialsFill volume per vial (µl)
Strand Switching Primer IISSPIIViolet120 µl
RT PrimerRTPYellow110 µl
cDNA RT AdapterCRTAAmber110 µl
Rapid Adapter TRAP TGreen110 µl
Annealing BufferABOrange110 µl
cDNA PrimercPRMWhite cap, grey label140 µl
Elution BufferEBBlack1500 µl
Short Fragment BufferSFBClear11,800 µl
Sequencing Buffer IISBIIRed1500 µl
Loading Beads IILBIIPink1360 µl
Loading SolutionLSWhite cap, pink label1400 µl
Flush BufferFBBlue61,170 µl
Flush TetherFLTWhite cap, purple label1200 µl

3rd party materials

Consumables

  • Agencourt RNAClean XP beads
  • Agencourt AMPure XP beads
  • Lambda Exonuclease (NEB, cat # M0262L)
  • NEBNext® Quick Ligation Reaction Buffer (NEB B6058)
  • T4 DNA Ligase 2M U/ml (NEB M0202)
  • 10 mM dNTP solution (e.g. NEB N0447)
  • LongAmp Hot Start Taq 2X Master Mix (NEB, M0533S)
  • Maxima H Minus Reverse Transcriptase (200 U/µl) with 5x RT Buffer (ThermoFisher, cat # EP0751)
  • RNaseOUT™, 40 U/μl (Life Technologies, 10777019)
  • USER (Uracil-Specific Excision Reagent) Enzyme (NEB, cat # M5505L)
  • Exonuclease I (NEB, M0293)
  • Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
  • Freshly prepared 70% ethanol in nuclease-free water
  • 1.5 ml Eppendorf DNA LoBind tubes
  • 0.2 ml thin-walled PCR tubes
  • Qubit RNA HS Assay Kit (ThermoFisher Q32852)
  • Qubit dsDNA HS Assay Kit (ThermoFisher Q32851)
  • Qubit™ Assay Tubes (ThermoFisher Q32856)

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
  • Qubit fluorometer (or equivalent for QC check)
  • Multichannel pipette capable of 20-200 μl

Compatibility

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

Kits

  • Flow Cell Wash Kit (EXP-WSH004)
  • Sequencing Auxiliary Vials (EXP-AUX110)

Flow cells

  • FLO-MIN106D
  • FLO-PRO002

Devices

  • MinION Mk1C
  • MinION Mk1B
  • GridION
  • PromethION