Assay Steps

 HS Library Preparation Kit

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The HS Library Preparation Kit protocol is a 3 days protocol that allows you to generate all ISS spots for the In Situ Sequencing.

Permeabilization

  • For Fresh Frozen tissue samples: with pepsin
  • For FFPE tissue samples: with Citrate buffer at 95-98 °C

Hybridization

Gene specific chimeric padlock probes are hybridized to the targeted RNAs. This requires an overnight incubation at 37°C.

Ligation of the chimeric padlock probes

This is an enzymatic reaction that requires 2 hours incubation at 37°C.

Rolling Circle Amplification

Ligated probes are locally amplified resulting in ISS spots containing multiple copies of the barcodes. This reaction requires an overnight incubation at at 30°C

Fluorescent Labeling

All ISS spots are visualized using an anchor fluorescent label
(Cy3, Cy5 or AF750). 
This requires a 30 minutes at RT.

Quality Control Imaging

Use a standard fluorescent microscope with a 20X objective to visualize the nuclei staining and the ISS spots.

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.