ISS KIT TECHNICAL NOTE

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The CARTANA In Situ Sequencing (ISS) Kit can sequence up to 600 genes in situ simultaneously with single cell resolution, with a high signal-to-noise ratio and higher quality reads than previous state-of-the-art in situ sequencing chemistry. This improved ISS chemistry leads to higher specificity during sequence analysis and decoding.

This technical note explains the CARTANA ISS chemistry and the CARTANA ISS Kit configuration.

ISS Kit key benefits:

• High signal-to-noise ratio

• Up to 600 genes identified simultaneously with single cell resolution

• High specificity & read quality

ISS KIT TECHNICAL NOTE

In Situ Sequencing (ISS) Kit

Figure 1: CARTANA ISS workflow. The CARTANA ISS Kit is applied to tissues prepared beforehand with the CARTANA HS Library Preparation Kit.

CARTANA In Situ Sequencing Workflow

Adapter probes hybridization

ISS spots sequencing

ISS imaging

2. In Situ Sequencing Kit

Transcript Y

Transcript X

Transcript Z

Transcript W

Chimeric padlock probes hybridization and ligation

HS Library Preparation Kit imaging

1 . HS Library Preparation Kit

Tissue permeabilization

Transcript X

Transcript Y

ISS spots generation and visualization

6x

3. In Situ Sequencing Data

ISS KIT TECHNICAL NOTE

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The CARTANA ISS Kit chemistry relies on adapter probes (AP) and a sequencing pool (SP) containing 4 different fluorescent labels (Alexa Fluor® 488, Cy3, Cy5 and Alexa Fluor® 750) that bind to the anchor and barcode sequences from the ISS spots generated during CARTANA HS Library Preparation (Figure 2). This produces a sequence-specific fluorescent signal within each ISS spot.

The new CARTANA ISS Kit chemistry is more robust and generates a stronger sequencing signal leading to a higher signal-to-noise ratio and higher specificity compared to the previous state-of-the-art ISS technology. Moreover, the new CARTANA ISS Kit chemistry allows for the decoding of up to 600 genes simultaneously.

Figure 2: Overview of a CARTANA ISS cycle. The adapter probe hybridizes to the anchor and barcode sequences inside the ISS spots in situ. The sequencing mix then binds to the complex, generating a sequence-specific fluorescent signal within each ISS spot. After imaging, the sequencing signal is removed from the ISS spots for the next sequencing cycle (6 cycles in total).

Figure 3: Example of visualization of 120 genes identified with the CARTANA ISS Kit in a mouse Fresh Frozen brain section (10µm-thick). Each colored symbol represents a unique targeted RNA. White: DAPI. A-C. At different zoom levels.

CARTANA In Situ Sequencing Kit Chemistry

CARTANA ISS Data

Adapter Probe

Sequencing Mix

Image

Remove Sequencing Signal from ISS spots

The raw data generated by the CARTANA ISS Kit consists of 20x images from 5 fluorescent channels (DAPI, Alexa Fluor® 488, Cy3, Cy5 and Alexa Fluor® 750) each taken as z-stack and flattened to 2D using maximum intensity projection.

After image processing and decoding, the results are summarized in a csv file (detailing the coordinates of each ISS spot). The analysis of the results represents each target RNA as a colored symbol (Figure 3). Refer to CARTANA ISS Data Technical Note for further information.

A. B.

2mm 300 µm 50 µm

C.

ISS KIT TECHNICAL NOTE

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Compared to the Allen Brain reference and to state-of-the-art ISS, the CARTANA ISS Kit shows a significant increase in read quality (Figure 4).

Thus, due to higher signal-to-noise ratio, CARTANA ISS chemistry is robust and consistent to spatially identify hundreds of transcripts , in one single tissue section.

Figure 4: A. Detection of Apoe, Gfap, Plp1 and NeuroD6 transcripts in mouse Fresh Frozen brain sections using state-of-the-art ISS chemistry versus the

new CARTANA ISS Kit (reference Allen Brain Atlas). B. Read quality comparison between state-of-the-art ISS and CARTANA ISS Kit chemistries.

Improved read quality of CARTANA ISS Technology

State of the Art

Apo

eG

fap

Plp

1N

euro

D6

CARTANA ISS Kit Allen Brain ReferenceA.

B.

ISS KIT TECHNICAL NOTE

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Sequencing cyclesThe CARTANA ISS Kit is used to decode barcodes from target-specific ISS spots generated during the in situ library preparation with the CARTANA HS Library Preparation Kit. Prior to decoding, the library preparation is validated through anchor labeling and imaging of all ISS spots. Decoding then starts with the hybridization of the 1st adapter probe (AP) pool (AP1, APMix 1) to the ISS spots and binding of the sequencing pool (SP) probes to the adapter probes (AP) (Figure 2).

After imaging, the sequencing signal is chemically removed to perform the next sequencing cycle. In total, 6 sequencing cycles are required for full decoding of up to 600 targets. The total reaction time for one sequencing cycle is approximately 2 hours, excluding imaging ( Figure 5).

DecodingAP pool hybridization leads to a target-specific ISS spot displaying a unique fluorescent signal in each cycle. Once all 6 sequencing cycles are analyzed, each ISS spot provides a unique fluorescent barcode identifying the targeted RNA.

In the example of decoding below (Figure 6): ISS spot X switches color at each sequencing cycle. After decoding, the color code of ISS spot X identifies transcript X.

In Situ Sequencing Procedure

Removal of previous probes ............. 10 (20) min

Adapter Probes Pool (AP) Mix .................. 60 min

Sequencing Pool (SP) Mix ...................... 30 min

Wash and mounting ................................ 10 min

Imaging .................................... Size dependent

X6

Figure 5: CARTANA ISS Kit protocol.

Figure 6: A brief schematic outlining CARTANA ISS Kit decoding method. The sequencing color within each ISS spot in each ISS cycle image is determined, yielding a color code for each targeted RNA. Each color code is then subjected to a transcript name search for the matching code (e.g. ISS cycle1: Cy5; ISS cycle 2: AF750; ISS cycle 3: Cy5; ISS cycle 4: Cy3; ISS cycle 5: AF488; ISS cycle 6: Cy5 = transcript X).

Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5 Cycle 6

Transcript X

Transcript X Transcript X Transcript X

Transcript Y Transcript Y Transcript Y

Transcript Z Transcript Z Transcript Z

Transcript W Transcript W Transcript W

…

…Cycle 2 Cycle 6Cycle 1

ISS KIT TECHNICAL NOTE

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Product configuration and requirements

ISS Kit content

Each CARTANA ISS Kit contains 9 tubes:

• 6 adapter probe pools (AP1-6) (one for each sequencing cycle)

• 1 sequencing pool (SP)

• Hybridization buffer (Buffer A)

• Blocking buffer (Buffer B)

The CARTANA ISS Kit is compatible with any HS Library Preparation Kit gene panel.

Tissue compatibility

• Fresh Frozen, Fixed Frozen and FFPE

• Any tissue, any species

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 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.

Image Analysis

• Cell profiler

• Fiji/ImageJ

• MATLAB

Contact Us

Technical Support: support@cartana.se

Information and Pricing: info@cartana.se

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