NanoString GeoMx DSP TMA-TNP Phase 4 WTA assay

FFPE slide preparation

Bake FFPE slides at 60°C for 1h 0m 0s .

Deparaffinize by sequential incubation with Xylene (0h 3m 0s twice), 100% EtOH (0h 1m 0s twice) and 95% EtOH (0h 1m 0s ).

Briefly rinse the slides with PBS and incubate with 1x Tris EDTA antigen retrieval buffer (9.0) for 0h 15m 0s (default condition for breast cancer tissue) at 100°C in a steamer.

Wash the slides with PBS for 0h 5m 0s (1 time).

place the slides in a coplin jar and expose RNA targets with Proteinase K at 0.1picomolar (pM) for 0h 15m 0s at 37°C in waterbath

Wash the slides with PBS for 0h 5m 0s (1 time) and proceed to the next step immediately

Preserve tissue morphology for soft tissues

Transfer the slides in a coplin jar containing 10% volume NBF (neutral buffered formalin) buffer and incubate them for 0h 5m 0s

Transfere the 10slides in a coplin jar containg NBF stop buffer (Tris base 24.5g and glycine 15g in water 2L ) and incubate them for 0h 5m 0s twice

Wash the slides with PBS for 0h 5m 0s

RNA target hybridization

Prepare hybridization solution with Buffer R (warm at Room temperature before opening) and thaw RNA detection probes on ice. Before use, mix thorouhgly by pipetting. Once thawed, refrigerate at 4°C for up to 3 months

Mix RNA probes at 1:10 dilution (eg. mix 25µL of RNA probe mix in 200µL of Buffer R and 25µL of DEPC-treated water to make up to final volume 250µL per slide )

Clean all equipment (especially for the hybridization chamber) with RNase AWAY and allow to dry or rinse with DEPC-treated water to avoid contamination

Wet Kimwipes with 2x SSC or DEPC-treated water and place them in the hybridization chamber to keep the slides from drying out

Add 200µL of hybridization solution to each slide and gently apply a Grace Bio-Labs hybriSlip onto the slide. Avoid the formation of air bubbles on the tissue

Incubate the slides in the hybridization chamber oven at 37°C for 16h 0m 0s (up to 24h 0m 0s )

Off-target probe removal

100% Formamide (FA) solution should be adjusted to Room temperature before preparing the stringent washing solution. Mix 4x SSC and 100% FA in equal volume and prewarm the solution in two coplin jars at 37°C in water bath

Gently remove the hybriSlip covers from the slides. Transfer slides in the stringent washing solution and wash twice at 37°C for 0h 25m 0s in water bath.

Wash slides with 2x SSC for 0h 5m 0s twice

Staining with morphological markers

Block the slides with Buffer W for 0h 30m 0s at Room temperature

Freshly prepare visualization markers (PanCK and CD45, Alexa-fluorescent conjugated) in 1:40 and Syto13 (1:10) dilution with Buffer W to a volume of 220ul per slide.

Incubate slides and visualization markers for 1h 0m 0s at Room temperature

Wash the slides with 2x SSC for 0h 5m 0s twice

Sample loading onto DSP GeoMx

Log onto GeoMx software and start with “New / Continue Run”.

After loading slides with the collection plate information, select (or create) the Readout group for NGS project. DSP is ready to scan slides.

A slide scan name is created and panel/visualization marker information is selected as below:

In the Probe Reagent Kit field, select Human NGS Whole Transcriptome Atlas RNA V1.0

1. Select the FITC/525 nm, Cy5/568 nm and Texas Red/615 nm channels.
2. For FITC/525 nm, select SYTO 13 as fluorophore, DNA as biological target and enter 50 as exposure time.
3. For Cy5/568 nm, select Alexa 532 as fluorophore, PanCK as biological target and enter 300 as exposure time.
4. For Texas Red/615 nm, select Alexa 594 as fluorophore, CD45 as biological target and enter 300 as exposure time.
5. Select FITC/525 nm as focus channel

When the scan area for each slide has been adjusted with sensitivity setting, select Scan .

ROI selection and AOI segmentation

After scanning is done, each color channel intensity is adjusted to show visualization markers along with tissue or cell line property

Each ROI is determined and selected by pathologist's guide, and drawn with circle (maximum 660um radius), rectangle (maximum 660x785um) or polygonal shape (maximum 660x785um). Due to the limitation of scan area in the slide loading slot, two same TMA slides were used to cover half of 88 cores in each slide (88 cores were embedded and slightly shifted to either left or right side of slide)

In the segment menu, 2 segmentation classes (Tumor, stromal and others) are added and parameters are set in the following order:

For Tumor segmentation (Segment 1), Alexa 532 (PanCK) is set to positive ("+") and the others set to ignore ("0") for the tumor collection

For stromal segmentation (Segment 2), Alexa 532 (PanCK), Alexa 594 (CD45) and FITC 525 (SYTO 13) were set to ignore ("0") to collect all other regions

Then click Generate Segments .

Once all segments are automatically generated, each channel parameter needs to be manually re-adjusted with pathologist's input to confirm if the segmentation is correctly done.

Caution : less than 20 cells in each segment is removed from collection due to threshold for low signal.

Once all AOI segmentation is complete, Exit Scan Workspace button icon is clicked to approve ROI selection and samples are collected in 96-well plate.

Sample sequencing

The sample collection plates were completely dried out at 65°C for 1h 0m 0s and shipped to the genomics core at Dana Faber Cancer Institute for NGS library preparation and sample sequencing

FASTQ raw sequencing files were generated and converted to DCC (Digital Count Conversion) files which can be imported to DSP for the analysis

QC DSP data and normalization

Select and queue the slides to analyze using "New Analysis" in the DSP server.

Determine the New Analysis file name and save it in the designated folder.

Open an analysis file and perform the QC with default parameters except Negative control (4 or lesser), No template PCR control (NTC count, 1,000-10,000) and minimum nuclei counts (50-100)

(Optional) QC passed samples are processed with Biological probe QC (only applying to CTA)

QC data were filtered with low expression across all segments. Choose 5-10% of segments frequency or higher of Limit of Quantification (LoQ) or both.

Upper Quartile (Q3) normalization from all target were performed after filtering out low expression targets (Q3 normalization method is recommended since larger number of targets may increase the noise of geomean normalized data) and data was exported in csv format