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cDNA-PCR Barcoding Kit V14

A sequencing kit optimised for identification and quantification of full-length transcripts with highest output, including barcodes for up to 24 samples.

This is an Early Access product

Product lead time: 6 weeks

This uses our latest Kit 14 chemistry.

SKU SQK-PCB114.24 Categorías ,
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Descripción

Information

This kit is recommended for users who:

  • have a limited amount of input material
  • wish to multiplex samples to reduce price per sample
  • 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 Barcoding Kit 24 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 – Barcoding (SQK-PCB114.24)
Multiplexing options • cDNA-PCR Barcoding Kit 24 V14 • 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

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.

Multiplexing samples onto one flow cell can reduce the cost per sample for a user. Below are some worked examples:

No barcodes 6 barcodes 12 barcodes 24 barcodes
Flow cell price $500 $500 $500 $500
Library price $99 $99 $99 $99
Price per sample $599 $104 $54 $28

The cDNA-PCR Barcoding Kit is used to prepare cDNA for nanopore sequencing for up to 24 samples, from an input of as low as 4 ng polyadenylated RNA. Users who do not have polyadenylated 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, with the incorporation of unique molecular identifiers (UMI) during this step. Taking full-length polyadenylated RNA, complementary strand synthesis and strand switching are performed using kit-supplied oligonucleotides. The kit contains 24 primer pairs which are used to generate and then amplify double-stranded cDNA by PCR amplification using primers that contain 5’ tags and facilitate the ligase-free attachment of Rapid Sequencing Adapters. Amplified and barcoded samples are then pooled together and the Rapid Sequencing Adapters are added to the pooled mix.

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 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 polyadenylated RNA, complementary strand synthesis and strand switching are performed using kit-supplied oligonucleotides. The kit contains 24 primer pairs that are used to generate and then amplify double-stranded cDNA by PCR amplification using rapid attachment barcode primers. These primers contain 5’ tags and facilitate 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.

PCB114.24 workflow

What’s in the box

The cDNA-PCR Barcoding Kit 24 V14 (SQK-PCB114.24) contains 24 unique barcodes and enough reagents for 6 reactions.

SQK PCB114.24 Kit content

Name Acronym Cap colour No. of vials Fill volume per vial (μl)
Strand Switching Primer II SSPII Violet 1 350
RT Primer RTP Yellow 1 200
cDNA RT Adapter CRTA Amber 1 200
Annealing Buffer AB Orange 1 200
Rapid Adapter RA Green 1 15
Adapter Buffer ADB Clear 1 100
Elution Buffer EB Black 2 500
Short Fragment Buffer SFB Clear 4 7,500
Sequencing Buffer SB Red 1 700
Library Beads LIB Pink 1 600
Library Solution LIS White cap, pink label 1 600
Barcode Primers 1-24 BP01-24 White 24 10
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, 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 Assay Kit(ThermoFisher, cat # Q32851)
  • Qubit™ Assay Tubes (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)
  • Multichannel pipette capable of 20-200 μl

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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