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Cas9 Sequencing Kit
SQK-CS9109

Targeted sequencing of genomic regions using CRISPR-Cas9

 Includes a Flow Cell Priming Kit
$625.00

Information

This kit is recommended for users who:

  • Want to target specific regions of 5-20 kb to high coverage (>100x) on a single MinION Mk 1B flow cell without PCR.
  • Want to sequence a larger, contiguous Region of Interest (ROI), using up to 100 target sites, in a single assay*
  • Have 1-10 µg of available high quality gDNA
  • Are interested in methylation patterns or other modified bases.
  • Have gene targets that are highly repetitive, or wish to evaluate the number of repeats in an expansion, where traditional amplification method or sequencing-by-synthesis methods could yield a biased result
  • Want to sequence long gene targets in a single pass that are not amenable to long-range PCR (>30 kb)

* Note: This is known as ‘tiling’ an ROI.

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.

Please also note that the Cas9 Sequencing Kit is compatible with R9.4.1 flow cells (FLO-MIN106D or FLO-MINSP6) only.

The Cas9 Sequencing Kit features:

FeatureProperty
Preparation time110 minutes
Input requirement1–10 µg of double-stranded DNA (5 µg recommended)
PCR RequiredNo
FragmentationNot required
Read lengthEqual to fragment length
Read type produced1D
Typical throughput★☆☆
Associated protocols/documentationCas9 Sequencing Kit (SQK-CS9109)
Associated analysis toolsCas9 Targeted Sequencing tutorial in EPI2ME Labs
Multiplexing optionsPlease contact Technical Support to discuss multiplexing
Pack size6 reactions
StabilityShipped 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:

Cas9 Targeted sequencing is a fast, flexible method for sequencing targets of interest without amplification. SQK-CS9109 offers users our protocol with the convenience and cost reduction of a kit.

Best coverage is achieved when using the CRISPR/Cas system to target a 5–20 kb region cutting twice at either end. For applications where only one end of the region is known, a single cut and read-out approach is possible.

For users that wish to target regions longer than 20 kb, we suggest a tiling approach and/or ensuring as highly intact starting DNA material as possible. It will not be possible to span larger regions without intact starting gDNA.

We strongly recommend starting with multiple probes and working out which approach works best for your experimental aims, DNA quality etc. Detailed recommendations can be found in the Targeted, amplification-free DNA sequencing using CRISPR/Cas document.

Licenses:

Oxford - Broad

Oxford - Caribou

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

The Cas9 Sequencing Kit offers a method of enriching regions of interest (ROI) from a genomic DNA sample. After DNA extraction, 5’ ends are dephosphorylated to reduce ligation of sequencing adapters to non-target strands. Cas9 ribonucleoprotein particles (RNPs), with bound crRNA and tracrRNA, are added to the genomic DNA, and bind then cleave the region of interest (ROI). dsDNA cleavage by Cas9 reveals blunt ends with ligatable 5’ phosphates. All of the DNA in the sample is dA-tailed, which prepares the blunt ends for sequencing adapter ligation. Sequencing adapters are ligated primarily to Cas9 cut sides, which are both 3’ dA-tailed and 5’ phosphorylated. The library preparation is cleaned up to remove excess adapters using AMPure XP beads and resuspended in Sequencing Buffer (Non-target molecules are not removed). The subsequent library preparation is added to the flow cell for sequencing.

Cas9 sequencing workflow

Protocols

Cas9 Sequencing Kit (SQK-CS9109)

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

What's in the box

The Cas9 Targeted Sequencing Kit contains sufficient reagents to generate six sequencing libraries.

SQK-CS9109 kit contents v2

NameAcronymCap colourNo. of vialsFill volume per vial (μl)
Adapter MixAMXGreen240
Ligation BufferLNBClear2200
Elution BufferEBBlack1200
Sequencing BufferSQBRed2300
L Fragment BufferLFBOrange21,800
S Fragment BufferSFBGrey21,800
Loading BeadsLBPink1360
Sequencing TetherSQTPurple110
PhosphatasePHOSBrown tube, yellow label150
TAQ PolymeraseTAQBrown tube, green label115
SPRI Dilution BufferSDBBrown tube, red label11,200
T4 DNA LigaseLIGBrown tube, blue label1140
dATPdATPBrown tube, grey label115
Reaction BufferRBBrown tube, orange label1180

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

  • S. pyogenes Cas9 Alt-R™ crRNAs (resuspended at 100 µM crRNA in TE pH 7.5)
  • S. pyogenes Cas9 tracrRNA (e.g., IDT Alt-R™, Cat # 1072532, 1072533 or 1072534) resuspended at 100 µM in TE pH 7.5 • Alt-R® S. pyogenes HiFi Cas9 nuclease V3, 100 µg or 500 µg (IDT, Cat # 1081060 or # 1081061)
  • Nuclease-free duplex buffer (IDT Cat # 11-01-03-01)
  • 1.5 ml Eppendorf DNA LoBind tubes
  • 0.2 ml thin-walled PCR tubes
  • Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
  • Agencourt AMPure XP beads

Equipment

  • Microfuge
  • Magnetic rack
  • 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

Optional equipment

  • Agilent Bioanalyzer (or equivalent)
  • Qubit fluorometer (or equivalent for QC check)
  • Eppendorf 5424 centrifuge (or equivalent)

Compatibility

The Cas9 Sequencing Kit can be used together with:

Kits

  • Flow Cell Wash Kit (EXP-WSH004)

Flow cells

  • FLO-MINSP6
  • FLO-MIN106D

Devices

  • MinION Mk1B
  • MinION Mk1C
  • GridION