The HS (High Sensitivity) Library Preparation Kit for In Situ Sequencing (ISS) uses a proprietary RNA probing technology to generate ISS libraries of up to 600 genes simultaneously in a tissue section at single cell resolution. The improvement over the previous cDNA-based Library Preparation Kit (NeuroKit) is the omission of the cDNA synthesis step and direct targeting of RNA transcripts. The HS Library Preparation Kit has a higher sensitivity compared to the previous cDNA based NeuroKit. Leading to highly efficient ISS on a variety of tissue preparations from all organs and in all species.
This technical note explains the principles of the HS Library Preparation
Kit product line.
HS Library Preparation Kit benefits:
CARTANA In Situ Sequencing Workflow
Figure 1: CARTANA ISS workflow. The CARTANA HS Library Preparation Kit is the first step of the workflow.
CARTANA chimeric padlock probe technology
CARTANA’s new HS Library Preparation Kit contains chimeric padlock probes that enable highly specific in situ library preparation of up to 600 different RNA sequences without the need for complementary DNA (cDNA) synthesis by reverse transcription (RT). Padlock probes are linear oligonucleotides comprised of 2 arms that are complementary to the targeted RNA sequence, and a non-hybridizing linker sequence containing an anchor sequence and the gene specific ISS barcode sequence (Figure 2A). Once both probe arms recognize and hybridize to their corresponding RNA sequence, they form a circle. The small gap between both arms is then closed by target-dependent ligation through a ligase that only connects both padlock probe arms when these fully match the target sequence, giving the reaction its high specificity. The closed circle is then copied locally by a DNA polymerase in a rolling circle amplification (RCA) reaction (Figure 2C). This generates an ISS spot of the detected RNA transcript that includes multiple copies of the ISS barcode sequence
corresponding to the detected gene. Any probe that does not hybridize to its target is not circularized, does not undergo RCA and is washed off the tissue or digested by exonuclease activity of the DNA polymerase during the RCA reaction.
Why chimeric padlock probes?
Chimeric padlock probes are predominantly comprised of DNA bases with the exception of the last base on the 3’ end that is a single RNA base (Figure 2A). This unique feature
allows the RNA-specific ligase to circularize the probe with much improved efficiency compared to normal DNA padlock probes hybridized to RNA, while maintaining the high specificity of a padlock probe ligation reaction (Figure 2B).
Thus, this newly developed chimeric probe strategy leads to increased sensitivity for the targeted RNAs within a panel.
Figure 2: Overview of the HS Library Preparation Kit chemistry. A. Chimeric padlock probes are linear DNA oligonucleotides with a terminal 3’ RNA base. B. When the arms find their matching RNA target within cells, the probe forms a circle that is sealed by the action of the RNA ligase. C. Circularized probes and their barcodes are copied by RCA, locally generating ISS spots (with gene specific barcodes) at the original position of the RNA target. D. All ISS spots are labeled with a general fluorophore, irrespective of their barcodes and E. visualized with fluorescent microscopy as bright dots to inspect the library preparation result. Pink/Magenta: ISS spots; Blue: DAPI staining.
Why direct RNA?
CARTANA’s previous version of the cDNA-based Library Preparation Kit (NeuroKit) uses regular padlock probes and a DNA ligase that requires a DNA target to circularize the
padlock probes. Therefore, RT of the RNA targets into cDNA is necessary. This step has low efficiency in situ, resulting in a significant loss of signal for every RNA molecule that is not transcribed. Moreover, the RT efficiency highly depends on the quality of both the tissue sample and RNA integrity, limiting the overall sensitivity and applicability of the NeuroKit.
By directly targeting RNA using chimeric padlock probes and a RNA ligase, it is now possible to apply HS Library Preparation Kit to a variety of tissue samples with different RNA quality
and fixation conditions. The new HS Library Preparation Kit works optimally on Fresh Frozen and FFPE tissue samples, even archived FFPE samples, which is critical for the analysis of clinical samples.
CARTANA HS Library Preparation Kit results
The CARTANA HS Library Preparation Kit generates ISS spots that can be 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 Cy7 channel, depending on choice of the labeling mix) together with the DAPI counterstaining of the nuclei. Images are obtained using a standard epifluorescence microscope with 20x objective (Figure 3).
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 their fluorescent intensity. However, the image of the Library Preparation 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.
Figure 3: 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.
Fresh Frozen and FFPE samples
We have thoroughly tested the HS Library Preparation Kit on both Fresh Frozen and FFPE samples. In Figure 4, comparison between Fresh Frozen and FFPE tissue sections in aorta samples shows that the HS Library Preparation Kit works optimally with both tissue preparation methods, providing a similar number and distribution of signals in both cases.
Figure 4: Performances of the HS Library Preparation Kit on Fresh Frozen and FFPE tissue samples. Detection of Rplp0 (low expressed housekeeping gene) in human Fresh Frozen (10 µm-thick) and FFPE (5 µm-thick) aorta tissue samples from the same patient. Green: Rplp0 ISS spots outlines, Gray: DAPI stain, scale bars: 1 mm. Tissue sections courtesy of Karin Lång & Flore-Anne Poujade, Karolinska Institute, Stockholm, Sweden.
All organs, all species
In addition, we have tested the HS Library Preparation Kit on a variety of FFPE samples from human, mouse and other species. Figure 5 illustrates the universality and performances of the HS Library Preparation Kit in mouse heart, lung, liver, kidney and small intestine samples.
The results confirm that the HS Library Preparation Kit is a robust and universal assay, enabling application of ISS in multiple research areas and in all species.
Figure 5: Performances of the HS Library Preparation kit on mouse FFPE tissue samples. Detection of Malat1 and Rplp0 (respectively high and low expressed housekeeping genes) in mouse heart, lung, liver, kidney and small intestine FFPE (5 µm-thick) tissue samples. Red: Malat1 ISS spots outlines, Green: Rplp0 ISS spots outlines, Gray: DAPI stain, scale bars: 1 mm.
Improved Performance of the HS Library Preparation Kit
We carefully assessed the sensitivity and specificity of the HS Library Preparation Kit.
The HS Library Preparation Kit is about 5 times more sensitive on Fresh Frozen samples and about 10 times more sensitive on FFPE samples, when compared to the previous cDNAbased Library Preparation Kit (NeuroKit) (Figures 6 and 7).
Figure 6: Performance of the HS Library Preparation Kit on FFPE tissue sections. Detection of NRAS, BRAF, CELF1, RPLPO on human melanoma FFPE serial tissue sections (5 µm-thick) with A. the cDNA-based Library Preparation Kit (NeuroKit) and B. the HS Library Preparation Kit. Symbols correspond to outlines of detected ISS spots each corresponding to a targeted transcript. Gray: DAPI, left panel scale bars: 2 mm and right panel scale bars: 200 µm. C. Comparison of the number of ISS spots/gene. Tissue sections courtesy of Carina Strell, Uppsala University, Uppsala, Sweden.
The maintained high specificity of the new HS library Preparation Kit was confirmed by comparing the gene expression patterns produced with HS Library Preparation and ISS Kits to those reported in the reference Allen Brain Atlas, gene by gene (Figure 7). The expression patterns are preserved and highly concordant with the classical in situ hybridization reference data.
The sensitivity of the new HS Library Preparation Kit has remarkably increased and now enables the accurate detection of low expressed transcripts. While boosting sensitivity, the high specificity is preserved, enabling optimal spatial analysis of mRNA, lncRNA, as well as splice variants.
Figure 7: Specificity of the HS Library Preparation Kit. A. Library preparation of a 14 gene panel on consecutive mouse brain Fresh Frozen sections (10 μm-thick) with the cDNA-based and the HS Library Preparation Kits. 1st panel: zoomed-out region with HS Library Preparation Kit, scale bar: 500 μm; 2nd and 3rd panels: zoomed -in same region images of ISS spot libraries prepared with HS Library Preparation Kit and cDNA-based Library Preparation Kit, respectively, scale bars: 50 μm. Blue: DAPI, white: anchor stain. B. Quantification of the total number of ISS spots/tissue section obtained with the cDNA-based and the HS Library Preparation Kit. Error bars correspond to standard deviation, n=3. C. Comparisons of selected gene expression patterns on mouse brain Fresh Frozen tissue sections (10 μm-thick) obtained with the HS Library Preparation Kit and compared to the Allen Brain Atlas reference. Colored symbols in the left panels, correspond to outlines of detected ISS spots for each gene transcript.
Improved Performance of the HS Library Preparation Kit
The general workflow of the HS Library Preparation Kit is very similar to that of the previous cDNA-based Library Preparation Kit (NeuroKit) (Figure 2).
As described above, the main difference is the absence of the RT step. The chimeric padlock probes are directly hybridized to the targeted RNA in an overnight incubation (Day 1, Figure 8). The second day, the excess and non-hybridized chimeric padlock probes are washed away and the ligation reaction is performed. Thereafter, circularized probes are amplified by RCA in a second overnight reaction (Figure 2C, Day 2 in Figure 8). This generates the ISS spot library (Figure 2D).
For an initial assessment of the HS Library Preparation Kit performance, ISS spots are labeled with one fluorophoretagged anchor probe oligonucleotide (in Cy3, Cy5 or Cy7 channel, depending on the choice of labeling mix) and counterstained with DAPI ( Day 3 in Figure 8). The ISS spot library is visualized with a fluorescence microscope, resulting in bright 0.5-1 µm dots surrounding the nuclei staining (Figure 2E and 3). The HS Library Preparation Kit procedure requires two days to be ready for the subsequent ISS sequencing cycles with the CARTANA ISS Kit (see CARTANA ISS Kit Technical Note) and ISS Data analysis (see CARTANA ISS Data Technical Note).
Figure 8: CARTANA HS Library Preparation protocol.
HS Library Preparation Kit content
Each kit contains 10 tubes:
- CARTANA Chimeric padlock probes (predefined and/or customized gene panel)
- Reaction mixes: RM1, RM2 and RM3
- Enzyme 1, Enzyme 2 and Enzyme 3
- Wash buffer 3 (WB3)
- Wash buffer 4 (WB4)
- Labeling mix (LM) (Cy3, Cy5 or AF750)
The optimization kit also contains two control probes (Malat1 and Rplp0) to validate the assay workflow and optimize the permeabilization conditions on specific samples.
- Fresh Frozen, Fixed Frozen and FFPE
- Any tissue, any species
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