Frequently Asked Questions

In Situ Sequencing Kit

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In Situ Sequencing Kit

At CARTANA, we have a ISS Starter Program. Every new user of the technology is enrolled in this program to start successfully with the technology. This program provides individual technical support based on your specific tissue samples and experimental design.

If you have any question, please contact support@cartana.se

 

What is the shelf life of the CARTANA products?

The CARTANA Library Preparation kit has a shelf life of 1 year (when reagents are stored in the recommended conditions).

The CARTANA probes have a shelf life of 2 years.

What are the imaging requirements?

For CARTANA ISS imaging, here are the 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 or 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.

Do you have recommended specifications for the high-throughput put slide scanner?

We do not have specific recommendations for high throughput scanner. It is important to follow the general requirements to image ISS results. Additionally, the scanner need to be able to perform z-stacks as ISS spots are localized in different planes of the sections.

For CARTANA ISS imaging, here are the requirements:

  • 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 or 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.

How do you plan to support core facilities and laboratories willing to set-up ISS?

As with the ISS Starter Program for the HS Library Preparation Kit, we have a program in place where we provide advice on microscope requirements, reference slides to optimize your imaging set-up, etc… In terms of analysis, advice, guidelines and training will be also provided.

How does CARTANA ISS compare to previous versions of ISS (SBL sequencing by ligation)?

The read quality of CARTANA ISS Kit is 20% higher than the read quality of the ISS-SBL.

We refer to the following publication:

Ke, et al. In situ sequencing for RNA analysis in preserved tissue and cells. Nature Methods (2013). DOI:10.1016/j.bbrc.2020.03.134

Can the method be performed on a programmed autostainer?

It is possible to automate the workflow on different staining instruments. For high-throughput, we recommend automating the parallel fluidic processing on slide stainers, such as Leica Bond or Thermo Fisher Labvision™, for which we have protocols available.

We are currently also working on developing automated systems to integrate with the microscope. But there are already published microfluidic setups that can be put together with off-the-shelf microfluidic components, which we are happy to point towards.

What is the purpose of the anchor sequence? Is the anchor sequence part of the barcode?

The anchor sequence is common to all probes and different than the gene-specific ISS barcode.

Below is a sketch of a chimeric padlock probe:

CARTANA Chimeric padlock probes design

Using a probe targeting the anchor sequence is used to visualize all ISS spots in the tissue samples, 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.

Imaging the library preparation results prior to the ISS procedure has two purposes:

  1. 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).
  2. The image of the HS Library Preparation is the reference to align the sequencing cycles during the ISS image processing steps.

How long does it take for sequencing 600 genes in 100 cells within a field of view with this new approach?

Since CARTANA ISS is a high throughput method, we do not calculate the time needed to perform ISS based on field of view with limited number of cells but on complete sample area. We consider one sample an area of 1 cm2

The total assay time is independent of the number of genes sequenced.

The HS Library Preparation Kit requires 3 days in total, with 2 overnight incubations:

CARTANA HS Library Preparation Kit Preparation time

The ISS Kit requires about 2 hours of hands-on and  1-6 hours per sequencing cycle:

CARTANA ISS Kit sequencing per cycle timeline

However time for imaging and analysis will vary depending on your imaging system and your data processing and analysis pipeline. , i.e. light source, filter cube exchanging speed, computing capacity etc…

We estimate that each sample (1 cm2) needs approximately one day for image processing and analysis.

Below is an overview of the high throughput capabilities of CARTANA ISS compared to other spatial genomics technologies:

CARTANA Throughput illustration

Is there a stopping point between imaging and sample processing during ISS?

Yes, it is possible to stop between imaging and sample processing between sequencing cycles. We recommend to stop no longer than 1 week between sequencing cycles (after imaging) for optimal performances.

Do you gain time if you reduce the number of genes you sequence?

No, performing the HS Library Preparation and ISS with a lower number of targets, does not reduce either the hands-on, nor the imaging time.

What is the resolution of CARTANA ISS?

The number of ISS spots that can be resolved depends directly on the microscope hardware settings. In-house, we use a 20x objective with 0.75 numerical aperture. ISS spots are between 0.5-1 µm big, so the question comes down to how many ISS spots can be resolved with that optical settings. An average number of a cell of 20 µm of diameter is in the 103 orders of magnitude.

When using 20X magnification, we estimate that we can detect 100-200 dots/ cells. This number increases to 500-1000 dots/cell when using 40X magnification.

What is the sensitivity of CARTANA ISS on single cells?

We estimate that CARTANA ISS can detect about 10% of all expressed transcripts in cells.

How exactly is the single cell resolution achieved?

For Fresh Frozen tissue samples, we recommend to use 10 µm thickness and for FFPE tissue samples 5 µm thickness ensuring a single layer of cells in the tissue section.

As CARTANA ISS an in situ technique, we sequence the mRNA transcripts directly in the tissues meaning that we can link each sequenced mRNA transcript to a specific cell.

 

How consistent are the transcript levels from different sections of the same sample?

CARTANA ISS is a reproducible assay. To improve consistency and reproducibility between samples, we highly recommend to prepare and process sections as one batch.

Reproducibility is good between CARTANA ISS experiments, but in order to merge data, some batch corrections might still be required.

 

Can we have a 3D resolution with CARTANA ISS?

We normally acquire z-stacks (3D info) since signals can be in different focal planes. However, for the analysis we do maximum intensity projections and only process 2D information.

 

How does the read quality differ between fresh frozen tissue and FFPE tissue?

In theory, when the HS Library Preparation has been perform optimally, there is no difference in read quality between Fresh Frozen and FFPE tissue samples. FFPE samples can present high autofluorescent background, which can affect read quality, but this is resolved by using an autofluorescence quenching step with TrueBlack during the HS Library Preparation protocol and at each sequencing cycle.