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cDNA-PCR Sequencing Kit V14

A sequencing kit optimised for identification and quantification of full-length transcripts with highest output.

This is an Early Access product

Product lead time: 6 weeks

This uses our latest Kit 14 chemistry.

  • Preparation time: 225 minutes + PCR
  • Read length: Enriched for full-length cDNA during PCR
  • Input amount: 10 ng poly(A)+ RNA or 500 ng total RNA
SKU SQK-PCS114 Categorías ,
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Descripción

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

This is an Early Access product. For more information about our Early Access programmes, please see this article on product release phases.

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 cDNA-PCR Sequencing Kit V14 features:

Feature Property
Preparation time 225 minutes + PCR
Input requirement 10 ng poly(A)+ RNA or 500 ng total RNA
RT Required Yes
PCR Required Yes
Read length Enriched for full-length cDNA during PCR
Kit chemistry Kit 14
Associated protocols cDNA-PCR Sequencing V14 (SQK-PCS114)
Multiplexing alternatives • cDNA-PCR Barcoding Kit V14 (SQK-PCB114.24)
• Flow Cell Wash Kit
Pack size 6 reactions
Stability Shipped 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

The cDNA-PCR Sequencing Kit is used to prepare cDNA for nanopore sequencing from an input of as low as 10 ng poly(A)+ RNA. Users who do not have poly(A)+ enriched RNA can use 500 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, with the incorporation of unique molecular identifiers (UMI) during this step. 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 kit has been upgraded to use our Kit 14 chemistry, which includes a reduction of free sequencing adapter and improved modal raw read sequencing accuracies (Q20+) with high output on our latest nanopore: R10.4.1. The flow cell priming and sequencing reagents are also included as part of the sequencing kit and have been reformulated to be compatible with the Kit 14 chemistry upgrade and the R10.4.1 nanopore. Other updates include fuel fix technology, allowing users to run longer experiments without the need for fuel addition during the run.

The cDNA-PCR Sequencing Kit also includes a 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 poly(A)+ tail lengths. Additionally, a new post-PCR wash step further reduces short artifacts that are seen when using low-quality samples.

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.

PCS114 workflow

What’s in the box

The cDNA-PCR Sequencing Kit V14 (SQK-PCS114) kit contains enough reagents for six reactions.

SQK PCS114 Kit content

Name Acronym Cap colour No. of vials Fill volume per vial (μl)
Strand Switching Primer II SSPII Violet 1 20
RT Primer RTP Yellow 1 10
cDNA RT Adapter CRTA Amber 1 10
Annealing Buffer AB Orange 1 10
Rapid Adapter RA Green 1 15
Adapter Buffer ADB Clear 1 100
cDNA Primer cPRM White cap, grey label 1 40
Elution Buffer EB Black 1 500
Short Fragment Buffer SFB Clear 1 1,800
Sequencing Buffer SB Red 1 700
Library Beads LIB Pink 1 600
Library Solution LIS White cap, pink label 1 600
Flow Cell Tether FCT Purple 1 200
Flow Cell Flush FCF Clear cap, light blue label 1 8,000

3rd party materials

Consumables:

  • Agencourt RNAClean XP beads (Beckman Coulter™, A63987)
  • Agencourt AMPure XP beads (Beckman Coulter™ cat # A63881)
  • Lambda Exonuclease (NEB, Cat # M0262L)
  • NEBNext® Quick Ligation Reaction Buffer (NEB, B6058)
  • T4 DNA Ligase 2M U/ml (NEB, M0202T/M)
  • 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, cat # 10777019)
  • USER (Uracil-Specific Excision Reagent) Enzyme (NEB, cat # M5505L)
  • Thermolabile Exonuclease I (NEB, Cat # M0568)
  • Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
  • Freshly prepared 70% ethanol in nuclease-free water
  • 5 ml Eppendorf DNA LoBind tubes
  • 2 ml thin-walled PCR tubes
  • Qubit RNA HS Assay Kit (ThermoFisher Q32852)
  • Qubit dsDNA HS Assay Kit (ThermoFisher, cat # Q32851)
  • Qubit™ Assay Tubes (Invitrogen, Q32856)

Equipment:

  • Hula mixer (gentle rotator mixer)
  • Magnetic separator, suitable for 1.5 ml Eppendorf tubes
  • Microfuge
  • Vortex mixer
  • Thermal cycler
  • Multichannel pipette
  • 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)

Compatibility

Kits:

  • Rapid Adapter Auxiliary V14 (EXP-RAA114)
  • Sequencing Auxiliary Vials V14 (EXP-AUX003)
  • RNA Control Expansion (EXP-RCS001)
  • Flow Cell Wash Kit (EXP-WSH004)
  • Flow Cell Wash Kit XL (EXP-WSH004-XL)
  • Flow Cell Priming Kit (EXP-FLP004)
  • Flow Cell Priming Kit XL (EXP-FLP004-XL)

Flow cells:

  • FLO-MIN114
  • FLO-PRO114M

Devices:

  • MinION Mk1B
  • MinION Mk1C
  • GridION
  • PromethION

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