Comprehensive Spatial Profiling of Species-agnostic Transcriptomes via Stereo-seq

Our research focuses on characterizing the ovarian tumor microenvironment in detail to identify predictive and therapeutic biomarkers. For example, we would like to find markers to predict chemo response and chemo resistance or novel therapeutical targets for second line-treatments. The experimental challenges include the sample handling, the quality assessment of the sample, and the length of the time for processing a sample.

While on the data analysis side, the challenges include things like the sparsity of the data, similar to other single cell processes or and also the mapping of areas, especially repetitive regions. After removing the stereo-sequencing N-Chip from its packaging, record the chip identification number. Without touching the N-Chip surface, clean the glass slide with 100%ethanol.

Using a micro pipette, carefully apply 10 microliters of ultraviolet curable ceramic resin around the edge of the stereo-sequencing N-Chip. Use a sealed foam swab to remove excess resin, leaving only a thin line around the chip edge. Place the slide into the ultraviolet curing device, ensuring the chip surface is not touched.

Now place the resin-applied chip on top of an opaque mask on the 405-nanometer ultraviolet light source to illuminate the back of the slide, and cure for five minutes. After curing, use sealed foam swabs soaked in 100%molecular-grade ethanol, then 70%ethanol, and finally, nuclease-free water to clean around the resin-applied edge. Submerge the chip three times in fresh nuclease-free water using a 50-milliliter conical tube.

Using compressed air, dry the chip with the full force of air at an angle of approximately 60 to 80 degrees, moving diagonally across the surface from about five centimeters away. Next, apply 150 microliters of Poly-L-Lysine solution evenly across the entire chip surface, and incubate the chip for 10 minutes at room temperature. Then remove the Poly-L-Lysine solution from the chip.

After the second wash, carefully dry the edges of the slide without touching the chip surface. Firmly dry the chip using compressed air as demonstrated earlier. While sectioning the FFPE tissue, do not touch the chip directly, and only cover 80%of the chip with tissue.

To prepare two microliters of single-stranded DNA staining solution in SSC buffer, dilute the Qubit single-stranded DNA reagent with 5X SSC buffer. Create a master mix of staining solution using a one to 20 dilution of ribonuclease inhibitor, a one to 200 dilution of the single-stranded DNA solution, and the remaining volume with 5X SSC buffer. Add 150 microliters of staining solution to each chip and incubate in the dark at room temperature for five minutes.

Then gently remove the staining solution from the corner of each chip using a pipette. After washing the chip twice with 0.1X SSC buffer for 10 to 15 seconds, carefully dry the edges of the slide. Add approximately three to five microliters of glycerol onto the slide to mount it.

Drop a coverslip from about one centimeter above the slide, keeping it level, and ensuring it does not touch the chip surface. Capture fluorescent images of single-stranded DNA-stained tissue using a wide-field microscope, ensuring 10%overlap between image tiles. Stitch the acquired images in image processing software such as ImageJ by first utilizing theoretical overlap to generate approximate tile positions, then applying pixel matching computational overlap to fine-tune the tiling.

Process the stitched images in StereoMap software, and confirm that image quality control passes before continuing. Submerge the stereo-sequencing slide in 30 milliliters of 0.1X SSC buffer until the coverslip detaches. Ensure the FFPE decrosslinking reagent is at room temperature and inspect it for particles.

Turn on the thermocycler and set it to 30 degrees Celsius for equilibration, 95 degrees Celsius for a 30-minute incubation, and infinite hold at four degrees Celsius. Assemble the cassette gasket and place the stereo-sequencing chip slide into the cassette. Then add FFPE decrosslinking reagent into the well of the stereo-sequencing slide cassette, apply sealing tape to the cassette and confirm it is tightly sealed.

Start the 30-minute incubation at 95 degrees Celsius. After 10 minutes, place 25 milliliters of methanol in a 50-milliliter container in a 20 degrees Celsius freezer, preparing about one milliliter per slide for later use. After removing the chip from the thermocycler, transfer the slide cassette to the bench and peel off the sealing tape.

Using a pipette, remove and discard the FFPE decrosslinking reagent from the cassette. Once the reagent has been completely removed, detach the cassette and gasket and discard them. Under a sterile fume hood, dry the edge of the slide, and apply a new silicone chamber if needed.

Add 500 microliters of chilled methanol from the 20 degrees Celsius freezer, ensuring the entire section is submerged. Prewarm PR solution on a 37 degrees Celsius dry block for 10 minutes before use. After fixation, in a fume hood, wipe off any excess methanol from the slide and wait for the remaining methanol on the chip to evaporate.

Assemble a new cassette end gasket. Once evaporation is complete, place the stereo-sequencing chip slide into the cassette. Add 200 microliters of prewarmed permeablization reagent to the chip.

Apply sealing tape to the stereo-sequencing slide cassette and ensure it is tightly sealed. Perform reverse transcription and then CDNA release and purification following the steps as shown here. Preheat nuclease-free water to 37 degrees Celsius.

Remove the seal from the chip. Pipette vigorously in each corner and in the center of the chip over the tissue without scraping or touching it. Collect all the complimentary DNA release mix from the chip and transfer it into a 1.5 or 2.0-milliliter centrifuge tube.

Add 350 microliters of preheated nuclease-free water directly onto the chip surface. Pipette vigorously with a smaller pipette, angling the tip to apply sheer force without touching the tissue or scraping the chip. Combine the wash with the 400 microliters of complimentary DNA release mix collected earlier, ensuring that only material from a single chip is combined.

Place 100 microliters of nuclease-free water on the chip. Seal the stereo-sequencing slide, and store it in the refrigerator until the end of the protocol. Single-cell segmentation using the SAW pipeline was achieved on FFPE sections, enabling mapping to non-polyadenylated RNAs such as tRNA, TRDMT1.

Interactive spatial visualization showed putative cell types using the proprietary software StereoMap, with clusters displayed in various colors across a zoomed-in region of the tissue. Whole tissue expression of the non-polyadenylated RNA TRDMT1 was visualized using StereoPi. The adaptations of the protocol address issues we had in full transcriptomics spatial profiling of fatty and fragile tissues by improving tissue adhesion, RNA accessibility, and handling for stereo-seqs on these difficult tissue types.

Stereo-seq currently provides higher resolution than other spatial transcriptomic methods with a resolution of 500 nanometers compared to two microns. It also provides unbiased capture needed for the study of the microbiome, viral, and non-polyadenylated transcripts.