Spatial Transcriptomics Protocol

Specimens should be handled wearing clean gloves treated with RNAse Away or with a clean RNAse Away treated forceps.

Cool Cryostat to -22°C. Clean the work surfaces with RNAse away and install a new cutting blade.

Place a clean small slide box inside the cryostat chamber to store slides containing freshly cut tissue.

Clean and treat the tissue holder will RNAse away and put it in the cryostat chamber . Adhere the OCT specimen to a tissue holder and allow it to equilibrate for a few minutes to reach the chamber temperature and strengthen the adhesion between the OCT block and the holder. This process can be aided via use of a heat extractor. The tissue holder was cleaned and treated with RNAse Away and cooled down.

In parallel, allow the cDNA capture slide (Visium product code #1000184) to equilibrate at -20°C.

Place the specimen in the cryostat with the tissue holder and cut at 10 µm thickness (1 section).

Try to avoid overlap with fiducials surrounding the capture area. If that is not possible, this can be adjusted informatically after sequencing if there is overlap. A minimum of 2 corners must be clear of tissue.

Store completed slides at -80°C in sealed 50mL RNAse free conical centrifuge tubes (Cat#91050). Use slides within 48 hours of cryosectioning.

Process specimens in batches of four because there are 4 capture areas per slide.

If tissue is folded or misplaced, the slide can be reset using Visium Spatial Slide Reset Protocol (CG000332 • Rev B) and reuse.

Use the Visium library preparation slide with affixed OCT sections and perform H+E staining.

Chill 40mL Methanol in a 50-ml centrifuge tube to -20°C.

Place a Thermocycler Adaptor on thermal cycler set at 37°C and equilibrate for 0h 5m 0s. Heating the thermal cycler lid is not required.

Place slide on the Thermocyler Adaptor with the active surface facing up and incubate 0h 1m 0s at 37°C. DO NOT close the thermal cycler lid, as the thermal cycler lid is normally heated to a higher temperature and it may touch the tissue sections when closed.

Completely immerse the slide in the prechilled methanol. Secure the tube cap. Incubate upright for0h 30m 0s at -20°C.

Remove slide from methanol and wipe excess liquid from the back of the slide without touching the tissue sections.

Add 500µL isopropanol to uniformly cover all tissue sections on the slide. Incubate 0h 1m 0s at -20Room temperature.

Drain reagent and air dry the slide for 0h 6m 0s.

Add 1mL Hematoxylin to uniformly cover all tissue sections on the slide. Incubate 0h 7m 0s at -20Room temperature.

Discard reagent and rinse slide sequentially in RNase free water. Wipe away excess liquid.

Add 1mL Bluing Buffer to uniformly cover all tissue sections. Incubate 0h 2m 0s at -20Room temperature.

Discard reagent and rinse slide sequentially in RNase free water. Wipe away excess liquid.

Add 1mL Eosin Mix to uniformly cover all tissue sections. Incubate 0h 1m 0s at -20Room temperature.

Discard reagent and rinse slide sequentially in RNase free water. Wipe away excess liquid.

Allow slide to air dry for 0h 6m 0s then incubate the slide on the Thermocycler Adaptor with the thermal cycler lid open for 0h 5m 0s at 37°C prior to imaging

After the specimen is fixed and stained with H+E reagents it is then imaged on a Keyence BZ-X810 scanning microscope at 10x or 20x resolution in the IU O’Brien Center for Microscopy.

Brightfield images of stained sections in the fiducial frames are collected as mosaics of 20x fields using a Keyence BZ-X810 microscope equipped with a Nikon 10X CFI Plan Fluor objective.

Including set-up time, 0h 24m 0s was required to image 4 sections. Image quality is assessed by visual inspection prior to RNA isolation and a given section can be re-imaged if necessary (~6 min needed).

Mosaics are stitched.

Bulk RNA quality is assessed on an adjacent section with an Agilent bioanalyzer prior to sectioning the tissue for spatial transcriptomics. Ideal RIN should be above 7, although some specimens may be able to yield adequate data with a RIN as low as 3.8.

Permeabilize stained tissue sections for 0h 12m 0s, mRNA was released to bind oligonucleotides on the capture areas with a test for RNA quality/quantity after permeabilization and isolation using real time PCR amplification from the Visium library prep slide.

cDNA synthesis - reverse transcription, second strand synthesis, denaturation, cDNA amplification, SPRIselect cDNA cleanup. cDNA quality is assessed by Agilent bioanalyzer.

Prepare and sequence the cDNA libraries on an Illumina NovaSeq 6000 with 28bp+120bp paired-end sequencing mode.

Using a Loupe Browser, map expression data to each “spot” and visualize overlying the histologic image