TITLE: GeoMX Digital Spatial Profiler Whole Transcriptome Assay  for Human Pancreas
AUTHORS: Ann  Fu, Yanping  Zhang, Heather  Kates, Jing  Chen, Alberto  Riva, Clayton E Mathews, Martha  Campbell-Thompson

[DESCRIPTION]
Purpose:
This protocol details processing fixed paraffin sections from human pancreas for the Nanostring GeoMX digital spatial profiler (DSP) whole transcriptome assay (WTA). This technology is based upon hybridization of samples with RNA reagents composed of >20,000 probes for full coverage of protein covering mRNA. The RNA probes are designed with complimentary RNA sequences and UV cleavable linkers and DSP barcodes. Morphology markers are used to identify cell types of interest with nuclear counterstaining permitting tissue spatial information. Sample regions of interest (ROI) are defined based on the 4 morphology markers and each ROI is illuminated with UV light thereby cleaving hybridized RNA probes. Cleaved probes are aspirated via microcapillary and deposited into a unique well of a 96-well collection plate. The collection plate is used for next generation sequencing (NGS). 

Scope:
This document was written following GeoMX- NGS guidelines (university.nanostring.com) with minor modifications for the University of Florida Molecular Pathology Core. 

The entire workflow is described for manual slide staining and GeoMX DSP instrument use. Steps for library construction, sequencing, bioinformatics, and data analysis are in outline form only. Library preparation and sequencing are outsourced to the University of Florida ICBR facility.  

Expected outcome:
RNA suitable for bulk RNA-sequencing will be obtained from fixed human pancreas sections within regions of interest and subareas defined by cell morphology markers.  

[BEFORE_START]
Preheat waterbath to 37 °C. 
Prepare 1x Antigen Retrieval Solution and preheat in steamer while slides are dewaxing.

[GUIDELINES]
Maintain the DSP instrument according to vendor recommendations including weekly wash buffer changes.
Maintain a RNAse-free work space.  
Validate morphology markers using assay conditions with antigen retrieval and RNA probe hybridization steps but mock RNA probe solution.  
Prepare all solutions using DEPC-water and RNAse-free supplies. 
Clean equipment with RNase AWAY, rinse with DEPC-treated water, and air-dry. 

[STEPS]
SECTION: Slide preparation
3. Bake slides at 60 °C for 60 min immediately before use.

SECTION: Slide deparaffinization
4. Dewax and rehydrate sections using slide tray stations or a Leica XL slide autostainer.  
Do not allow tissue sections to dry once rehydrated and avoid touching tissue when drying around edges. 
SECTION: Slide deparaffinization
4.1. Xylene 3 x 5 min. CitriSolv or another xylene substitutes can be used.
SECTION: Slide deparaffinization
4.2. 100% Ethanol 2 x 5 min 

SECTION: Slide deparaffinization
4.3. 95% Ethanol 2 x 5 min 

SECTION: Slide deparaffinization
4.4. PBS 1 x  1 min 

SECTION: Antigen retrieval
5. Prepare Antigen Retrieval Solution (1x Tris EDTA, pH 9) in Coplin jar. 
Pre-heat in steamer.

SECTION: Antigen retrieval
5.1. Transfer slides to Antigen Retrieval Solution jar and steam for 20 min. 

SECTION: Antigen retrieval
5.2. Immediately transfer slides to PBS. 

SECTION: RNA retrieval
6. RNA retrieval solutions needed:
Proteinase K working solution: Place in Coplin jar and prewarm to 37 °C for 15 min in water bath.
NBF stop solution in two Coplin jars
PBS wash 
SECTION: RNA retrieval
6.1. Incubate slides in Proteinase K jar at 37 °C for 15 min in water bath. 

SECTION: RNA retrieval
6.2. Wash PBS 1 x  5 min. Proceed to next step immediately.

SECTION: RNA retrieval
7. Post-retrieval fixation for pancreas 
SECTION: RNA retrieval
7.1. Incubate in 10% NBF for 5 min.

SECTION: RNA retrieval
7.2. Incubate in NBF stop buffer 2 x 5 min. 

SECTION: RNA retrieval
7.3. Wash PBS 1 x 5 min.

SECTION: RNA probe hybridization and stringency washes
8. Prepare RNA probe mixture in dedicated RNAse-free area away from nCounter work or other NGS workflows. 
SECTION: RNA probe hybridization and stringency washes
8.4. 
 
     Buffer R      RNA Probe Mix      DEPC H20      Final Volume        (200ul x n)  _________         (37.5 ul x n)  + ________      (12.5 ul x n)  + ________      (250 ul x n)  = ________   
 
SECTION: RNA probe hybridization and stringency washes
8.1. Warm buffer R to RT before opening vial.
SECTION: RNA probe hybridization and stringency washes
8.2. Thaw RNA Probe Mix on ice. Before use, mix thoroughly by pipetting.   Store unused RNA detection probes at 4 °C up to 6 months or re-freeze.

SECTION: RNA probe hybridization and stringency washes
8.5. Prepare slide hybridization incubator with RNAse AWAY and dry or rinse with DEC water. Place Kimwipes on bottom and wet with 2 x SCC or DEPC water to create humid environment during overnight hybridization.
SECTION: RNA probe hybridization and stringency washes
8.7. Remove each slide from PBS, tilt and wipe off excess PBS, then place in slide holder of hybridization chamber. 
Work quickly to avoid tissue drying.
SECTION: RNA probe hybridization and stringency washes
8.8. Immediately add 200 ul RNA probe solution to each slide. Do not introduce air bubbles during pipetting.
SECTION: RNA probe hybridization and stringency washes
8.9. Apply a Hybrislip over each section by gently laying down over tissue, starting from one edge, and avoiding introduction of air bubbles.
SECTION: RNA probe hybridization and stringency washes
8.10. Repeat steps 8.5-7 for each slide.
Close slide tray, insert into hybridization oven, and clamp in place. 
SECTION: RNA probe hybridization and stringency washes
8.11. Incubate for  960 min at 37 °C. Slides must remain under humid conditions to prevent drying out of hybridization solution. The chamber can be placed in a ziplock bag for additional security.
SECTION: RNA probe hybridization and stringency washes
9. Post-hybridization stringency washes.
Solutions needed: 
2xSSC
2xSSC-T
2x SSC/50% formamide 
SECTION: RNA probe hybridization and stringency washes
9.1. Warm 100% formamide to RT before opening. 
Prepare 2x SSC/50% formamide by mixing 100ml each of 4x SSC and 100% formamide. 
Add solution to 2 Coplin jars and preheat to 37 °C in water bath .
SECTION: RNA probe hybridization and stringency washes
9.2. Dip slides in 2x SSC to remove coverslips. 
If they do not come off immediately, move to 2x SSC-T for maximum of  5 min. 
If coverslip have not come off in 2x SSC-T, move slides to stringent washes where coverslips should come off.
SECTION: RNA probe hybridization and stringency washes
9.4. Wash in 2xSSC/50% formamide 2 x 25 min at 37 °C  in water bath. 

SECTION: RNA probe hybridization and stringency washes
9.5. Wash in 2xSSC 2 x 5 min. 

SECTION: Morphology marker incubation
10. Incubate sections with up to 3 morphology markers (MM) and one DNA counterstain to define cell types of interest. Morphology markers are validated by in-house testing using the RNA assay conditions. 
Clean humidity chamber with RNAse AWAY.

SECTION: Morphology marker incubation
10.2. Remove one slide from 2xSSC and tap clean on stacked clean towels to remove excess buffer.
Cover section with 200ul Buffer W for 30 min in dark slide chamber while preparing morphology marker solution. Make sure Buffer W extends 2-3 mm past section borders.
SECTION: Morphology marker incubation
11. Tap slide edge on towels to remove MM solution then wash slides in 2x SSC 2 x 5 min.  
SECTION: GeoMX DSP run
12. Start run by selecting New/Continue Run under Data Collection in the GeoMx DSP Control Center.
SECTION: GeoMX DSP run
12.1. Clean the bottom of slide with 70% ethanol and place slide in slide holder with slide label towards front of instrument.
SECTION: GeoMX DSP run
12.2. Complete loading slides then lower slide tray clamp. Cover sections with 6 ml Buffer S. Record slots for each slide. 
SECTION: GeoMX DSP run
13. Follow prompts in the GeoMx DSP Control Center to identify slides. 
SECTION: GeoMX DSP run
13.1. Name slides according to protocol https://dx.doi.org/10.17504/protocols.io.n2bvj6dnblk5/v1 
Include UF HuBMAP sampleID and morphology marker abbreviations in order of channels followed by date (MMDDYY). Example: P1-21_DNA_PanCK_INS_CD31_121021.
SECTION: GeoMX DSP run
13.2. Upload unzipped configuration files to DSP kits used for Morphology and WTA kits used in slide preparations (nanostring/dsconfigfiles). Config files must match names of assays used on slides.
SECTION: GeoMX DSP run
13.3. Define scan area for each sample using x- and y-sliders and scan. The minimum tissue slide filters particles and small tissue areas from scan. The sensitivity slider adjusts intensity of tissue identification.

SECTION: GeoMX DSP run
13.4. Draw regions of interest (ROI) using geometric, segmented or other tools. 
Maximum RO1 size is ~   diameter circle. 

SECTION: GeoMX DSP run
13.5. Segment ROIs for AOI (area of illumination) for cell types of interest based on positive or negative selection by morphology markers. Minimum of ~100 nuclei/AOI is recommended for sufficient RNA. 

Insulin is used to designate beta-cells in the endocrine compartment, CD31 is used to define endothelium, and panCK is used to define ductal epithelium in the exocrine compartment. Acinar cells are defined by the lack of staining for CD31 and PanCK in the exocrine ROI. 
SECTION: GeoMX DSP run
13.6. Exit scan workspace and approve ROI to start ROI collection. 120 min 

SECTION: Data analysis
19. Share FASTQ files and annotation file with data analyst who will analyze RNAseq data using R scripts for GeoMx DSP workflow (Bioconductor - GeoMxWorkflows). Data analysis may also be performed with GeoMX DSP software on auxiliary server.   
SECTION: Data analysis
19.2. Run various testing including QC, data scaling, normalization, background, statistical tests, and ratios. 
SECTION: Data analysis
19.1. Manage annotations (tags). Add columns to further define regions and cell types from morphology markers.
SECTION: Data analysis
19.3. Analyze in spatial context using spatial deconvolution software and scRNAseq data.
SECTION: Data analysis
19.4. Complete analysis and upload data to HuBMAP HIVE.
SECTION: NGS library and sequencing
18. Core facility will process Illumina FASTQ sequencing files and transfer back to laboratory. 
SECTION: GeoMX DSP run
13.7. After ROI/AOI collections are completed, follow prompts to remove slide holder and collection plate.
SECTION: GeoMX DSP run
12.3. Load a 96-well aspiration plate in DSP. Check solution containers are filled and waste container is empty.
SECTION: GeoMX DSP run
13.8. Remove buffer from each slide with pipette and dispose. Open clamps and unload slides. Store slides in 2x SSC at 4 °C up to 7 days. 
SECTION: NGS library and sequencing
17. Core facility will perform Illumina sequencing.
SECTION: Slide preparation
2. Cut paraffin block to fresh sample. 
Section at   and place sections centrally on Superfrost Plus slides as shown in Fig. 1. Tissue samples should have maximum width and length of ~14 and 36 mm, respectively, for inclusion within the GeoMX instrument slide holder gasket. Tissue outside the gasket region should be carefully removed without creating folds or leaks to gasket seal. 
Air dry mounted slides overnight before user or storage. 
Recommended storage is for < 2 weeks in a desiccator at 4 °C.
  
1. This procedure details processing of paraffin slides, in set of 4,  for GeoMX WTA by manual methods. Methods using the Leica BOND autostainer are detailed on the Nanostring website.
Paraffin blocks containing 4% paraformaldehyde fixed human pancreas are screened by H&E staining and histopathology review https://www.protocols.io/view/human-pancreas-histopathology-assessment-n92ld956xg5b/v1.

Wash steps in this protocol may be abbreviated such as one wash= 1 x and three washes= 3 x. 
Room temperature (RT) is used except where different temperature is noted.
SECTION: Morphology marker incubation
10.1. 
Thaw nuclear marker, vortex then spin down for at least 1 minute to pellet any precipitates. Pipette from top of vial only. Close tightly after use and refreeze at -20 °C. 
SECTION: Morphology marker incubation
10.3. Prepare MM solution. Mix solution by flicking followed by quick spin down.
 
 
 
           Nuclear Marker  MM1  MM2  MM3  Buffer W  Total Volume    Slide Number  22 ul x n  5.5 ul x n  5.5 ul x n  5.5 ul x n  187ul x n  220 ul x n    1  22  5.5  5.5  5.5  187  220    2  44  11  11  11  374  440    3  66  16.5  16.5  16.5  561  660    4  88  22  22  22  748  880  
 
SECTION: Morphology marker incubation
10.4. 
Working with one slide at a time, remove Buffer W by tapping slide edge on stacked towels then add 200ul morphology marker solution/slide in humidity chamber. Repeat for each slide.
SECTION: Morphology marker incubation
11.1. Submerge slides in 2x SSC for 2 x 5 min. Load slides in DSP within 360 min of last wash for optimal results.
SECTION: GeoMX DSP run
13.9. Clean slide holder as directed and replace in instrument. Follow prompts to close instrument door.
SECTION: Slide reuse
14. RNA assay slides can be restained following UV exposure for 3 minutes to strip tags from bound probes followed by washes using with 2xSSC-T. Proceed with additional staining.

SECTION: NGS library and sequencing
15. Transfer collection plate to the University of Florida ICBR Gene Expression and Genotyping Core or other NGS core facility for library preparation and Illumina sequencing.  
Refer to Nanostring user manual for additional information. 
SECTION: NGS library and sequencing
16.1. Library will be evaluated for quality and fragment size distribution using a BioAnalyzer 2100 (Agilent).
SECTION: NGS library and sequencing
16. Library will be prepared after indexing PCR with pooling and purification.
SECTION: GeoMX DSP run
13.10. Fill in the GeoMX Seq Code plate to be used with the plate barcode in the Readout Group Information grid. 
Click update button and finalize and download readout package buttons to USB drive. 
SECTION: Antigen retrieval
5.3. Wash slides in PBS 1x 5 min. 
SECTION: Antigen retrieval
5.4. Slides can be stored in PBS up to 60 min.
SECTION: RNA retrieval
7.4.  Slides can be stored in PBS wash solution up to 60 min at RT or up to 6 hrs at 4 °C.
SECTION: RNA probe hybridization and stringency washes
8.3. Make RNA probe hybridization solution as per Table 1. Before use, briefly vortex and quick spin down.
8.6. The hybridization chamber can be a key point of contamination by oligo probes.
SECTION: RNA probe hybridization and stringency washes
9.3. Do not force off coverslips to avoid tissue damage.
SECTION: RNA probe hybridization and stringency washes
9.6. After last wash, slides can be stored in 2xSSC for up to 60 min.
SECTION: RNA probe hybridization and stringency washes
9.7. Carefully wash all containers coming into contact with the hybridization solution (Proteinase K, formamide mixture 2XSSC) with RNAse AWAY to prevent probe contamination. Use separate Coplin jars for different probe mixtures.
SECTION: Morphology marker incubation
10.5. Incubate slides in dark slide chamber for 60 min 
SECTION: Morphology marker incubation
11.2.  Alternatively, store slides in 2xSSC for up to 2 days at 4 °C in dark.
SECTION: Morphology marker incubation
11.3. Slides must be stored in dark to avoid cleavage of DSP tags by UV light.