Imaging the CARTANA HS Library Preparation Kit results is the first step in the CARTANA data analysis procedure.
The library preparation generates ISS spots which are fluorescently labeled with the labeling mix (included in the HS Library Preparation Kit). All ISS spots in the tissue samples are visualized with one general fluorescent label, irrespective of their barcodes (in Cy3, Cy5 or Alexa Fluor® 750 channel, depending on the choice of labeling mix) together with DAPI counterstaining of the nuclei. Images are obtained using a standard epifluorescence microscope with 20x objective (Figure below).
Imaging the library preparation results prior to the ISS procedure has two purposes:
- To validate the library preparation using the number of ISS spots and the fluorescent intensity of ISS spots.
However, the image of the sequencing library does not reveal which transcript an ISS spot represents (this is done during the ISS procedure).
- The image of the HS Library Preparation is the reference to align the sequencing cycles during the ISS image processing steps.
Library Preparation result images using an epifluorescence microscope. Tissue: mouse Fresh Frozen brain section (10 µm-thick) Blue: DAPI, white: anchor stain. A. Fully zoomed-out image, scale bar: 1 mm, B. zoomed-in image with low and C. high contrast, scale bars: 50 µm.
For an overview on CARTANA ISS image analysis and ISS data decoding and processing please download CARTANA In Situ Sequencing technical note.