Visium 10X spatial seq

The first part of both the Visium Fresh Frozen and FFPE workflow is to test the RNA integrity of your tissue. This step is highly important to secure high quality spatial data. In brief, tissue is sectioned, followed by RNA extraction using and Qiagen Rneasy kit and finally the RNA quality is assessed on an Agilent Tapestation.

Step 1: Cryo/Microtome Sectioning (CFIM, Day 1)

The OCT or FFPE embedded tissue of interest is sectioned into a pre-cooled Eppendorf tube for RNA extraction.

Step 2: RNA Extraction (User lab., Day 1)

After sectioning, RNA is extracted from the tissue sections using a Qiagen Rneasy mini or FFPE kit. Please consult the Visium tissue prep. guide and the Rneasy protocol for further information. This step is carried out at your own laboratory.

Step 3: RNA Quantification and Integrity Control (FCSC, Day 1-2)

Following RNA extraction, the RNA conc. Is quantified on a Qubit fluorometer and analyzed on an Agilent Tapestation to assess the RNA integrity number (RIN, fresh frozen) or degree of fragmentation (DV200, FFPE). If the RNA passes the quality threshold (RIN≥7 or DV200≥ 50%) you can proceed to the Visium Tissue Optimization workflow (Fresh Frozen) (item 2 on the list) or directly to the FFPE Visium Spatial Gene Expression Workflow (item 4 on the list).

The purpose of the Visium Tissue Optimization workflow is determine the optimal permeabilization time for your specific tissue type. The optimal permeabilization time, is crucial for achieving high quality data and should be determined every time you run a new type of tissue.

Step 1: Cryo-sectioning (CFIM, Day 1)

In this step, the given tissue is Cryo-sectioned and seven tissue sections transferred to the Visium Spatial Tissue optimization Slide. Slides with mounted tissue sections can be stored at -80°C for up to 4 weeks.

Step 2: Fixation, Staining and Imaging (CFIM, Day 2)

Following cryo-sectioning the tissue on the Visium tissue optimization slide needs to be methanol fixated, H&E or IF stained and optionally imaged. Note, that the optimal permeabilization may differ between H&E and IF stained tissues.

Step 3: Tissue Permeabilization and Fluorescent cDNA synthesis (FCSC, Day 2)

The fixed and stained tissue sections on the Visium tissue optimization slide are permeabilized over a specific time course, with seven different time points. The permeabilization releases mRNA from the cells in the tissue. The mRNA is captured by dT-oligoes on the slide followed by fluorescent cDNA synthesis.

Step 4: Fluorescent imaging of the cDNA (CFIM, Day 2)

To determine the optimal permeabilization time, the capture areas of the Visium Tissue Optimization slide are scanned using a fluorescent scanning microscope. The optimal permeabilization time is then applied during the Visium Spatial Gene Expression workflow.

Proceed to item 3 on the list.

Following RNA quality control and tissue optimization, you can proceed onto the Visium Spatial Gene Expression workflow for fresh frozen samples.

 Step 1: Cryo-sectioning (CFIM, Day 1)

In this step, the given tissue is cryo-sectioned and placed on to the capture areas of the Visium spatial gene expression slide. Slides with mounted tissue sections can be stored at -80°C for up to 4 weeks.

Step 2: Fixation, Staining and Imaging (CFIM, Day 2)

Following cryo-sectioning the tissue on the Visium spatial gene expression slide needs to be methanol fixated, H&E or IF stained and imaged.

Step 3: Tissue permeabilization, cDNA synthesis and Library preparation (FCSC, Day 2)

Tissue sections on the Visium spatial gene expression slide are then permeabilized, releasing the mRNA from the cells in the tissue. The mRNA is captured by barcoded dT-oligoes on the capture area of the Visium spatial gene expression slide. The mRNA is the reversed transcribed to cDNA, which in turn is used as input for NGS library preparation.

Step 4: Quality control, Quantification and Sequencing of NGS Libraries (FCSC, Day 3-4)

The quality of the prepared Visium NGS libraries is assessed using an Agilent Tapestation, confirming the correct fragment sizes and that there are no adapter dimers present. The libraries are then quantified by qPCR and pooled for sequencing on the Illumina Novaseq 6000.

Following RNA Quality control of your FFPE Tissue, you can proceed to the Visium Spatial Gene Expression workflow for FFPE samples.

Step 1: Microtome-sectioning (CFIM, Day 1)

In this step, the given tissue is microtome-sectioned and placed on to the capture areas of the Visium spatial gene expression slide. Dried slides with mounted tissue sections can be stored at room temperature in a desiccator for up to 2 weeks.

Step 2: Deparaffinization, Staining, Imaging and Decrosslinking (CFIM, Day 2)

Following microtome-sectioning the tissue on the Visium spatial gene expression slide needs to be, Deparaffinized, H&E stained, imaged and finally decrosslinked. If the tissue needs to undergo IF staining, the tissue is first deparaffinized, decrosslinked, then IF stained and finally imaged.

Step 3: Probe hybridization, ligation, capture and NGS library prep. (FCSC, Day 2-3)

Following step 2, a mouse or human whole transcriptome probe panel is added to the tissue on the Visium spatial gene expression slide. The panel consists of a probe pair for each transcript target. Following target hybridization, the probe pairs are ligated, released from the tissue and captured by barcoded dT oligoes on the Visium slide. The captured probes are thene extended, generating barcoded probe products for NGS library preparation.

Step 4: Quality control, Quantification and Sequencing of NGS Libraries (FCSC, Day 3-4)

The quality of the prepared Visium NGS libraries is assessed using an Agilent Tapestation confirming the correct fragment sizes and that there are no adapter dimers present. The libraries are then quantified by qPCR and pooled for sequencing on the Illumina Novaseq 6000.