TITLE: GeoMx-NGS Manual RNA Slide Preparation Protocol
AUTHORS: Angela R.S.  Kruse, Morad C Malek, Jamie Allen, Melissa Farrow, Jeff Spraggins

[DESCRIPTION]
This protocol describes the preparation of tissue samples for spatial transcriptomics via the Nanostring GeoMx Digital Spatial Profiler. 

Expected outcome: Tissue samples probed with RNA probes that are ready for subsequent spatial sampling. 

[GUIDELINES]
Be sure to clean all surfaces with RNase-away to prevent cross-contamination with RNA probes or RNA degradation. 

[STEPS]
SECTION: PREPARE REAGENTS
1. 
 
          Reagent  Dilution  Storage    95% EtOH  Prepare   500 mL of 95% ethanol by adding 25 mL of DEPC- treated   water to 475 mL of 100% ethanol. Change at least weekly.  RT    1X PBS   pH 7.4  Prepare 1 L of 1X PBS by combining 100 mL of 10X PBS and    900 mL of DEPC-treated water. Don't reuse.  4°C    10%   neutral buffered formalin (NBF)  Caution: NBF is hazardous, handle with care and minimize inhalation   risks. Work with 10% NBF in the fume hood.    OK to reuse.  RT    Proteinase   K  Prepare 1 µg/mL by adding 10 μL of 20 mg/mL Proteinase K to 200 mL of   1X PBS made with DEPC-treated water    Note: Should be   prepared fresh daily; don't reuse. Take care to pipette accurately.   Inaccurate concentration of Proteinase K will affect assay performance.   Serial dilutions are recommended.  RT    NBF stop   buffer  Prepare 24.5 g Tris base and 15 g Glycine to 2 L DEPC- treated water.    Do not reuse. Solution will lose efficacy with   repeated use.  RT    2X SSC  Prepare 1 L of 2X SSC by combining 100 mL of 20X SSC and 900 mL of   DEPC-treated water. Do not reuse.  RT    2X SSC-T  Prepare 100 mL of 2X SSC by combining 10 mL of 20X SSC, 1 mL of 10%   Tween-20, and 89 mL of DEPC-treated water. Do not reuse.  RT    4X SSC  Prepare 1 L of 4X SSC by combining 200 mL of 20X SSC and 800 mL of   DEPC-treated water. Do not reuse.  RT  
 


SECTION: Prepare Tissue Samples
2. Unstained  tissue  sections  should  be 5 µm thick on Superfrost Plus slides. Tissue sections must be placed in the Scan Area (the green area in the slide diagram) in the center of the slide and be no larger than 36.2 mm long by 14.6 mm wide. They should not overlap the slide gasket or the Tip Calibration area (this is the triangular region to the left of the green scan area in the slide diagram). If sections are larger than this size and/or placed off-center, the tissue located outside the Scan Area will not be scanned by the GeoMx DSP instrument.
 
SECTION: Prepare Tissue Samples
3. Bake sections on slides in a 60°C drying oven for a minimum of 30 minutes prior to deparaffinization. Longer baking times may be necessary for some tissues to adhere to the slide; this should be empirically tested. If tissue falls off, then baking longer could help.
SECTION: Deparaffinize and rehydrate FFPE tissue sections (31 minutes)
4. You will need the following items/reagents for this step: Steamer, Staining jars, 1X Tris EDTA
(pH 9.0), DEPC-treated water, Citrisolv/Xylene, 100% EtOH, 95% EtOH, and 1x PBS. See
the Equipment, Materials, and Reagents list for more details.


SECTION: Deparaffinize and rehydrate FFPE tissue sections (31 minutes)
5. Fill the steamer reservoir up to the fill line with water. Place two staining jars inside, one containing 1X Tris EDTA (pH 9.0) and one containing DEPC-treated water (use sufficient volume to cover slides up to the label). Loosely cover each jar with aluminum foil instead of the jar lid; this allows for a thermometer reading in a later step. Preheat the steamer to 100°C. 

The Nesco steamer takes 1 hour to heat the jars to a stable maximum temperature near 100°C. Final temperature can be checked by inserting a digital thermometer through the hole in the lid of the steamer .
SECTION: Deparaffinize and rehydrate FFPE tissue sections (31 minutes)
6. Preheat the water bath to 37°C.
SECTION: Deparaffinize and rehydrate FFPE tissue sections (31 minutes)
7. Deparaffinize and rehydrate FFPE tissue sections. Place the slides in a rack and gently perform the following washes using staining jars (see Figure 4).

 
Ensure you have sufficient buffer to cover all slides in container for the washes below. After the last wash, slides can be stored in the 1x PBS for up to one hour.
SECTION: Perform target retrieval (15 - 25 minutes)
8. You will need the following items/reagents for this step: Steamer, Staining jars, 1X Tris EDTA
(pH 9.0), DEPC- treated water (all pre- heated in the previous step) and 1X PBS . See the
Equipment, Materials, and Reagents list for more details.


SECTION: Perform target retrieval (15 - 25 minutes)
9. Without removing the lid, place an instant-read digital thermometer through the vents in the steamer lid and pierce the aluminum foil covering the Tris EDTA. Ensure the Tris EDTA has reached ~99°C. Reset the steamer's timer to ensure consistent heating during incubation. 
SECTION: Perform target retrieval (15 - 25 minutes)
10. Once the Tris EDTA has reached the appropriate temperature, carefully remove the steamer lid and jar covers. Dip the slides into the DEPC-treated water for 10 seconds; this step assists in maintaining the temperature of the retrieval buffer. Quickly transfer the slides to the Tris- EDTA.
SECTION: Perform target retrieval (15 - 25 minutes)
11. Incubate the slides. Incubation times and temperatures may differ by tissue and may need to
be empirically determined. The table below lists starting conditions (see Table 5). If the tissue
type you wish to use is not listed, start with the default conditions: 15 min @ 100°C.

  

SECTION: Perform target retrieval (15 - 25 minutes)
12. When the timer is finished, move slides to room temperature PBS immediately.
SECTION: Perform target retrieval (15 - 25 minutes)
13. Wash in room temperature PBS for 5 minutes Slides can be stored up to 1 hour in the PBS.
SECTION: Expose RNA targets (10 - 30 minutes)
14. You will need the following items/reagents for this step: Staining jars, water bath, Proteinase K,
and 1X PBS. See the Equipment, Materials, and Reagents list for more details.

SECTION: Expose RNA targets (10 - 30 minutes)
15. Dilute Proteinase K. Proteinase K concentration and incubation times differ by tissue and may
need to be empirically determined. Modifications for certain tissues listed below (see Table 6). If
the tissue type you wish to use is not listed, start with the default conditions: 1 μg/mL for 15
min. Note that Proteinase K solution must be diluted fresh immediately before use. 

  

SECTION: Expose RNA targets (10 - 30 minutes)
16. Place proteinase K in a Coplin jar and warm it to 37°C in water bath.
SECTION: Expose RNA targets (10 - 30 minutes)
17. Incubate slides in proteinase K at 37°C using the time and concentration or tissue- dependent conditions listed in the table above (see Table 6).

SECTION: Expose RNA targets (10 - 30 minutes)
18. Wash in 1X PBS for 5 minutes. Proceed to the next step immediately.
SECTION: Postfix—Preserve tissue morphology for soft tissues (20 min)
19. You will need the following items/reagents for this step: Staining jars, 10% NBF, NBF Stop
Buffer and 1X PBS. See the Equipment, Materials, and Reagents list for more details.
SECTION: In Situ Hybridization (overnight)
21. You will need the following items/reagents for this step: Hybridization chamber, hybridization
oven, Buffer R, RNA Probe Mix, and DEPC-treated water. See the Equipment, Materials, and
Reagents list for more details.

Prepare buffers: Warm Buffer R to room temperature (RT) before opening. Thaw RNA detection probes on ice. Before use, mix thoroughly by pipetting. Once thawed, do not re-freeze probes. Refrigerate at 4°C for up to 3 months.

Prepare the hybridization chamber according to product instructions. The hybridization ovens listed in the Equipment section are not light-permeable. If your chamber is light-permeable, minimize light exposure (e.g., by wrapping the lid in aluminum foil).
SECTION: In Situ Hybridization (overnight)
23. Clean all equipment with RNase AWAY and allow to dry or rinse with DEPC-treated water (see Important note, above). The hybridization chamber can be a key source of contamination by oligos. Arrange fresh Kimwipes on bottom of the chamber. 
SECTION: In Situ Hybridization (overnight)
24. Wet the Kimwipes with 2X SSC or DEPC-treated water. Take care that the Kimwipes and 2X SSC do not contact the slides. Hybridization solution can wick off of the slides if in contact with Kimwipes or liquid. Kimwipes should be thoroughly damp, but standing buffer should not be present.
SECTION: In Situ Hybridization (overnight)
25. One at a time, remove slides from PBS, wipe away excess liquid, and set in hybridization chamber. Take care not to let the slides dry out.
SECTION: In Situ Hybridization (overnight)
26. Add 200 μL hybridization solution to each slide. Take care not to introduce any bubbles.
SECTION: In Situ Hybridization (overnight)
22. Make hybridization solution (n= number of slides) (see Table 7).
If adding custom targets to the RNA assay, add the appropriate volume of custom RNA spike-in
(up to two, see below). Add DEPC-treated water if needed to bring volume up to the final
volume.
  

SECTION: Postfix—Preserve tissue morphology for soft tissues (20 min)
20. Post-fix the tissue by performing the washes listed in the figure below (see Figure 7).

 
Slides can be stored in the final PBS wash up to 1 hour at RT or up to 6 hours at 4°C. 

SECTION: In Situ Hybridization (overnight)
27. Gently apply a Grace Bio-Labs HybriSlip. Start by setting one edge of the coverslip down in solution on the slide, then gradually laying down the rest of the coverslip to avoid the formation of air bubbles.
SECTION: In Situ Hybridization (overnight)
28. Repeat steps 4–6 for each slide.
SECTION: In Situ Hybridization (overnight)
29. Close hybridization chamber, insert into oven, and clamp into place. Incubate at 37°C overnight (16–24 hours).
SECTION: Perform stringent washes to remove off-target probes (90 minutes)
30. You will need the following items/reagents for this step: Water bath, 4X SSC, and formamide.
See the Equipment, Materials, and Reagents list for more details.
SECTION: Perform stringent washes to remove off-target probes (90 minutes)
31. Before you begin, ensure water bath is set to 37°C for later use. Warm 100% formamide to room temperature before opening. Once formamide is at room temperature, make Stringent Wash by mixing equal parts 4X SSC and 100% formamide. Fill two staining jars with Stringent Wash and preheat them in 37°C water bath. 
SECTION: Perform stringent washes to remove off-target probes (90 minutes)
32. Dip the slides in 2X SSC-T and allow the coverslips to slide off by themselves. Minimize the time in SSC-T; continue to the wash steps within 5 minutes. Slides with coverslips that have not come off after 5 minutes can be moved to stringent wash. Coverslips should come off in the first stringent wash.
SECTION: Perform stringent washes to remove off-target probes (90 minutes)
33. Perform the washes listed in the figure below (see Figure 11). After the last wash, slides can be stored in 2X SSC for up to one hour.
  

SECTION: Add morphology markers (100 minutes)
34. You will need the following items/reagents for this step: Humidity chamber, Buffer W, nuclear
stain (SYTO 13), morphology markers, and 2x SSC . See the Equipment, Materials, and
Reagents list for more details.
Before you begin: Remove nuclear stain (SYTO 13) from the freezer (stored at -20°C) and allow to
warm to room temperature on bench.

IMPORTANT: Before using the humidity chamber in the following steps, clean it with RNase
Away. Prep the humidity chamber by lining with Kimwipes wetted with 2x SSC.


SECTION: Add morphology markers (100 minutes)
35. Block with Buffer W. Move one slide at a time to the tray. Transfer tray to the humidity chamber for antibody staining. Cover tissue with up to 200 μL Buffer W and leave at RT for 30 minutes. Cover to prevent light exposure. 

Ensure adequate Buffer W surrounds the edges of the tissues so they don't dry out. Use a pipette tip to gently move the solution so there is a 2-3 mm border around the tissue. A hydrophobic barrier can be used at this step, but must be carefully removed with a razor blade after antibody incubation.
SECTION: Add morphology markers (100 minutes)
36. Picofuge thawed nuclear stain (SYTO 13) for at least 1 minute to bring the solution to the bottom of the vial and precipitate insoluble particles. Do not pipette from the bottom of the vial. Close all vials tightly and store the remaining nuclear stain (SYTO 13) at -80°C.
SECTION: Add morphology markers (100 minutes)
37. Prepare morphology marker solution (n = number of slides)(see Table 8). Prepare up to 220 μL per slide.

  
*If a different number of detection Ab or morphology marker tubes are used, Buffer W amount needs to
be adjusted to bring total volume up to 220 μL per slide.


SECTION: Add morphology markers (100 minutes)
38. Spin morphology marker solution and antibodies by flicking and spinning down.
SECTION: Add morphology markers (100 minutes)
39. Remove Buffer W by tapping slide onto a Kimwipe.


SECTION: Add morphology markers (100 minutes)
40. Cover tissue with antibodies (up to 200 μL). Stain for 1 hour in the humidity chamber at RT. Cover to prevent light exposure.
SECTION: Add morphology markers (100 minutes)
41. Wash 2 times for 5 min in 2x SSC.

When the slides are prepared, load them immediately on the GeoMx DSP (see the GeoMx DSP
Instrument User Manual). 

If needed, slides can be stored at 4°C in 2xSSCT for 6 hours or 2xSSC for one week. 
SECTION: Add morphology markers (100 minutes)
42. Once GeoMx DSP aspirates are collected, the collection plate may be stored at -20°C until ready for library preparation.