Checklist

In Situ Sequencing Kit

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The lists all the material and reagents required to perform experiments with the CARTANA ISS Kit.

ISS Kit Content

Product Product name Part Number P/N

(small ISS Kit P/N 3110-01)

Part Number P/N

(large ISS Kit P/N 3110-02)

Adapter Probe Pool 1 AP 1 3229-01 3229-02
Adapter Probe Pool 2 AP 2 3230-01 3230-02
Adapter Probe Pool 3 AP 3 3231-01 3231-02
Adapter Probe Pool 4 AP 1 3232-01 3232-02
Adapter Probe Pool 5 AP 2 3233-01 3233-02
Adapter Probe Pool 6 AP 3 3234-01 3234-02
Sequencing Pool SP 3128-01 3128-02
Buffer A Buffer A 3127-01 3127-02
Buffer B Buffer B 3126-01 3126-02

Material & Reagents not Supplied in the Kit

  • Incubator/Oven: optimal temperature 37°C. Temperature needs to be stable, and preferably monitored with a verified external thermometer.
  • Humidity chambers: a slide chamber that prevents evaporation for extended periods of incubation up to 45°C (see General Guidelines below).
  • PAP pen: Recommended ImmEdge Pen (Vector Labs, Cat. No H-4000) is the only PAP pen compatible with the ISS protocol. Refer to D082 HS Library Preparation Kit Tips and Guidelines for more information.
  • Absolute Ethanol: 70%
  • Formamide: 100%
  • TrueBlack: Recommended TrueBlack® Lipofuscin Autofluorescence Quencher (Biotium, Cat. No 23007).
  • Wash Buffer 1 (WB1): 01% DEPC treated PBS (room temperature overnight, then autoclaved). See D082 HS Library Preparation Kit Tips and Guidelines for more information.
  • Wash Buffer 2 (WB2): 01% DEPC treated PBS-Tween 0.05% (room temperature overnight, then autoclaved). See D082 HS Library Preparation Kit Tips and Guidelines for more information.
  • Mounting Medium: Recommended SlowFade™ Gold Antifade Mountant (ThermoFisher Scientific, Cat. No S36936).
  • Coverslips: compatible with the objective used for optimal imaging.

Imaging requirements

  • Epifluorescence microscope with autofocus function.
  • Automated (x, y, z) stage with the stage controller.
  • Light source and filter cubes for optimal imaging of DAPI (358/461), Alexa Fluor® 488 (495/519), Cy3 (550/570), Cy5 (650/670) and Alexa Fluor® 750 (749/775)
  • Objectives 4X, 20X and 40X.
  • Digital camera (pixel size 5-7 µm)
  • PC and data storage with specifications suitable for imaging and processing data ranging from 4TB to 60TB.

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.