Checklist

High Sensitivity Library Preparation Kit

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The lists show all the material and reagents required to perform CARTANA HS Library Preparation on Fresh Frozen, Fixed Frozen or FFPE tissue samples.

HS Library Preparation Kit content

Product Product name Part Number P/N

(small Kit P/N 1110-01)

Volume*

(µL)

Part Number P/N

(large Kit P/N 1110-02)

Volume*

(µL)

Reaction Mix 1 RM1 1131-01 480 1131-02 1440
Reaction Mix 2 RM2 1133-01 555 1133-02 1665
Reaction Mix 3 RM3 1117-01 540 1117-02 1620
Enzyme 1 Enzyme 1 1112-01 15 1112-02 45
Enzyme 2 Enzyme 2 1134-01 30 1134-02 90
Enzyme 3 Enzyme 3 1135-01 60 1135-02 180
Wash Buffer 3 WB3 1130-01 600 1130-02 1800
Wash Buffer 4 WB4 1132-01 600 1132-02 1800
Labeling Mix* LM* 1019-01, 1029-01, 1036-01 600 1019-02, 1029-02, 1036-02 1800
Probes** Probes** Mouse 4101-01

Human 4101-01

120 Mouse 4101-02

Human 4101-02

360

* depending on product configuration

** for probes targeting different species, please contact CARTANA

The HS Library Preparation Kit also contains two control probes (Malat1 and Rplp0) to validate the assay workflow and optimize the permeabilization conditions on specific samples.

Material & Reagents not Supplied in the Kit

  • Incubator/Oven: temperature 60°C to bake the FFPE slides.
  • Incubator/Oven: temperature range: 30°C – 37° Temperature needs to be stable, and preferably monitored with a verified external thermometer.
  • Steamer or hot plate: for Target Retrieval step.
  • Humidity chambers: a slide chamber that prevents evaporation for extended periods of incubation up to 37°C (see General Guidelines below).
  • SecureSeal™ chambers: (Grace Biolabs). It is highly recommended to use SecureSeal™ chambers if proceeding to ISS after sample preparation. Different chamber volumes can be used that suits the tissue size or experimental objective (see SecureSeal Hybridization Chamber Guidelines v1.0 (D117)).
  • Pap pen: (ImmEdge, Vector Labs) only recommended for sample quality testing – not leading up to ISS. See HS Library Preparation Kit Tips and Guidelines (D082) for more information.
  • Fixation solution (FS): 37% Formaldehyde (Sigma Aldrich 252549). Make aliquots from a fresh bottle and store these at -20°C. Thaw and dilute only before use and discard leftovers. See HS Library Preparation Kit Tips and Guidelines (D082) for more information.
  • Permeabilization Solution (PS):1 M HCl in DEPC treated MQ H2O (0.01% DEPC, room temperature overnight, then autoclaved). See HS Library Preparation Kit Tips and Guidelines (D082) for more information.
  • Pepsin: Pepsin (Sigma Aldrich P7012). Always prepare fresh in Permeabilization Solution (PS).
  • Target Retrieval: Citrate buffer pH 6.0 (Sigma Aldrich C9999). Always prepare fresh in DEPC treated MQ H2O
  • Wash Buffer 1 (WB1): 01% DEPC treated PBS (room temp overnight, then autoclaved). See HS Library Preparation Kit Tips and Guidelines (D082) for more information.
  • Wash Buffer 2 (WB2): 01% DEPC treated PBS-Tween 0.05% (room temperature overnight, then autoclaved). HS Library Preparation Kit Tips and Guidelines (D082) for more information.
  • DEPC treated MQ H2O:01% DEPC treated MQ H2O (room temperature overnight, then autoclaved).
  • Xylene or xylene substitute: 100% for deparaffinization.
  • Ethanol: 70% and 100%.
  • Mounting Medium: Recommended: SlowFade™ Gold Antifade Mountant (ThermoFisher Scientific, Cat. No S36936).
  • Coverslips: any compatible with fluorescence microscopy.
  • Fluorescence microscope: 2.5x – 20x (preferably) objective and filters for DAPI and Cy3 or Cy5 or AF750.
  • General labware: Eppendorf tubes, slide holders/Coplin jars/beakers, etc.

It is recommended to work in an RNase free environment to avoid degradation of RNA molecules. Always use fresh RNAse free reagents for samples preparation.

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