TITLE: NanoString GeoMx DSP TMA-TNP Phase 4 WTA assay
AUTHORS: Jinho Lee, Gabriel Zangirolani, Koei Chin, Heidi S Feiler, Christopher Corless

[DESCRIPTION]

This protocol outlines the NanoString GeoMx Digital Spatial Profiler Whole Transcriptome Atlas (DSP WTA) assay that was applied in the Human Tumor Atlas Network (HTAN) Tissue MicroArrary (TMA) -TransNetwork Project (TNP).

The TMA-TNP evaluates various characterization and analytics methodologies on a large array of breast tumor samples representing a broad spectrum of disease state and subtype. A commercially available anonymized breast tumor TMA was purchased and serial sections were distributed.  Participating HTAN Centers characterized the FFPE specimens using various imaging platforms and generated a spatially resolved cell type/state census using each center’s method of choice. Data was recorded in a common repository to enable joint analysis.

The protocol that immediately precedes this one for TMA-TNP Phase 4  can be found at:  dx.doi.org/10.17504/protocols.io.ewov1o7wolr2/v1. It describes FFPE block serial sectioning, slide processing and TMA sample distribution. Two compartments (Tumor and Stroma) in each TMA core were analyzed to determine cell-to-cell interactions in the tissues.

[STEPS]
SECTION: FFPE slide sample preparation
1. FFPE slide preparation
SECTION: FFPE slide sample preparation
1.1. Bake FFPE slides at 60 °C for 60 min .

SECTION: FFPE slide sample preparation
1.2. Deparaffinize by sequential incubation with Xylene (3 min  twice), 100% EtOH (1 min twice) and 95% EtOH (1 min ).

SECTION: FFPE slide sample preparation
1.3. Briefly rinse the slides with PBS and incubate with 1x Tris EDTA antigen retrieval buffer (pH 9.0) for 15 min (default condition for breast cancer tissue) at 100 °C  in a steamer.

SECTION: FFPE slide sample preparation
1.4. Wash the slides with PBS for 5 min (1 time).


SECTION: Postfixation
2. Preserve tissue morphology for soft tissues



SECTION: Postfixation
2.1. Transfer the slides in a coplin jar containing 10 % volume NBF (neutral buffered formalin) buffer and incubate them for 5 min 

SECTION: Postfixation
2.2. Transfere the 10slides in a coplin jar containg NBF stop buffer (Tris base 24.5 g  and glycine 15 g  in water 2 L ) and incubate them for  5 min twice 

SECTION: Postfixation
2.3. Wash the slides with PBS for 5 min 

SECTION: in situ hybridization
3. RNA target hybridization
SECTION: in situ hybridization
3.1. 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

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

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

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

SECTION: Stringent washes
4. Off-target probe removal
SECTION: Stringent washes
4.1. 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

SECTION: Stringent washes
4.2. Gently remove the hybriSlip covers from the slides. Transfer slides in the stringent washing solution and wash twice at 37 °C  for 25 min  in water bath.

SECTION: Stringent washes
4.3. Wash slides with 2x SSC for 5 min twice

SECTION: Sample loading to GeoMx
6. Sample loading onto DSP GeoMx
SECTION: Sample loading to GeoMx
6.1. Log onto GeoMx software and start with “New / Continue Run”. 
SECTION: Tissue staining
5. Staining with morphological markers


SECTION: Tissue staining
5.1. Block the slides with Buffer W for 30 min at Room temperature 


SECTION: Tissue staining
5.2. 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.

SECTION: Tissue staining
5.3. Incubate slides  and visualization markers for 60 min at Room temperature 

SECTION: Tissue staining
5.4. Wash the slides with 2x SSC for 5 min twice

SECTION: Sample loading to GeoMx
6.4.  When the scan area for each slide has been adjusted with sensitivity setting, select Scan.
SECTION: Sample loading to GeoMx
6.3. 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
Select the FITC/525 nm, Cy5/568 nm and Texas Red/615 nm channels.
For FITC/525 nm, select SYTO 13 as fluorophore, DNA as biological target and enter 50 as exposure time.
For Cy5/568 nm, select Alexa 532 as fluorophore, PanCK as biological target and enter 300 as exposure time.
For Texas Red/615 nm, select Alexa 594 as fluorophore, CD45 as biological target and enter 300 as exposure time. 
Select FITC/525 nm as focus channel 

SECTION: Sample loading to GeoMx
6.2. After loading slides with the collection plate information, select (or create) the Readout group for NGS project. DSP is ready to scan slides.
SECTION: in situ hybridization
3.6. Incubate the slides in the hybridization chamber oven at 37 °C for 960 min (up to 1440 min )

SECTION: in situ hybridization
3.5. Add 200 µL 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

SECTION: FFPE slide sample preparation
1.6. Wash the slides with PBS for 5 min (1 time) and proceed to the next step immediately
SECTION: FFPE slide sample preparation
1.5. place the slides in a coplin jar and expose RNA targets with Proteinase K at 0.1 picomolar (pM) for 15 min at 37 °C in waterbath


SECTION: QC and data normalization
9.4. (Optional) QC passed samples are processed with Biological probe QC (only applying to CTA)  

SECTION: QC and data normalization
9.3. 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)  
SECTION: QC and data normalization
9.2. Determine the New Analysis file name and save it  in the designated folder.
SECTION: QC and data normalization
9.1. Select and queue the slides to analyze  using "New Analysis"  in the DSP server.
SECTION: QC and data normalization
9. QC DSP data and normalization
SECTION: ROI selection and sample collection
7. ROI selection and AOI segmentation
SECTION: ROI selection and sample collection
7.1. After scanning is done, each color channel intensity is adjusted to show visualization markers along with tissue or cell line property

SECTION: ROI selection and sample collection
7.2. 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)

SECTION: ROI selection and sample collection
7.3. 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.

SECTION: ROI selection and sample collection
7.4. 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. 

SECTION: ROI selection and sample collection
7.5. 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.
SECTION: NGS library preparation and sequencing
8. Sample sequencing 
SECTION: NGS library preparation and sequencing
8.1. The sample collection plates were completely dried out at 65 °C for 60 min and shipped to the genomics core at Dana Faber Cancer Institute for NGS library preparation and sample sequencing


SECTION: NGS library preparation and sequencing
8.2. FASTQ raw sequencing files were generated and converted to DCC (Digital Count Conversion) files which can be imported to DSP for the analysis

SECTION: QC and data normalization
9.5. 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. 
SECTION: QC and data normalization
9.6. 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