TITLE: snPATHO-seq
AUTHORS: Tony Wang, Michael Roach, Jasmine Plummer, Luciano G Martelotto

[DESCRIPTION]
Formalin-fixed paraffin-embedded (FFPE) samples are valuable but under-utilised in single-cell omics research due to their low DNA and RNA quality. Leveraging recent single-cell genomic technology advances, we introduce snPATHO-seq: a versatile method to derive high-quality single-nucleus transcriptomic data from FFPE samples.

[STEPS]
SECTION: Paraffin removal
1. Cut 1 or 2 approximately 25 μm-thick sections (punches are also possible) and transfer to a 1.5 mL Eppendorf tube. 

 

SECTION: Paraffin removal
2. Add 1 mL and incubate for 10 min at Room temperature or 50-55 °C 

 

SECTION: Rehydration
5. 
 

Add 1 mL and incubate for 1 min at Room temperature, then carefully remove ethanol without disturbing the sections/punches.

SECTION: Rehydration
10. Add 1 mL, incubate for 1 min at Room temperature , carefully remove RPMI1640 wash
SECTION: Tissue dissociation
12. Digest tissue for 45-60 mins(*) at 37°C in a Thermomixer at 800 RPM.
  

 

SECTION: Tissue dissociation
11. Prepare  1 mL:
1 mL 
0.25-1 mg/mL 
0.25-1 mg/mL 
0.25-1 mg/mL 
1 U/uL 

Add this 1 mL to the sections/punches.

 

SECTION: Lysing the cells
13. 
 

Add 400 µL to the sample and mix by inverting 5 times, then centrifuge 850 rcf, 5 min, 4 °C 
SECTION: Rehydration
6. Repeat step 5: add 1 mL, incubate for 1 min at Room temperature, remove ethanol wash
SECTION: Rehydration
7. Add 1 mL, incubate for 1 min at Room temperature, remove ethanol wash
SECTION: Rehydration
8. Add 1 mL, incubate for 1 min at Room temperature, remove ethanol wash
SECTION: Rehydration
9. Add 1 mL, incubate for 1 min at Room temperature, remove ethanol wash
SECTION: Paraffin removal
3. Carefully remove Xylene without disturbing the sample.
SECTION: Paraffin removal
4. Repeat steps 2 and 3 two more times:
Add 1 mL, incubate 10 min at Room temperature, remove xylene
Add 1 mL, incubate 10 min at Room temperature, remove xylene

SECTION: Lysing the cells
14. Prepare 2 mL as follows:
2 mL
2 % (v/v)
1 U/uL 

SECTION: Lysing the cells
16. Homogenize the sample using a douncer/pestle by stroking 10-20 times (or as needed).
SECTION: Lysing the cells
17. Add a further 750 µL
SECTION: Lysing the cells
18. Homogenize by pipetting using a P1000 pipette (10 times), then incubate on ice for 5 min 

SECTION: Lysing the cells
19. Repeat step 18: pipette 10 times and incubate on ice for 5 min 
SECTION: Lysing the cells
20. Optional but very useful when possible: If the dissociation and disaggregation look 
almost complete (i.e. only very small chunks of undigested tissue or fat are visible to the naked eye) gently pass the sample through a 25G needle for 20 times (avoid foaming). It is essential to ensure that no large chunks remain before passing though needle. If large chunks or fat remain the needle will definitely block, so just skip this step. This optional step will increase the nuclei release.

SECTION: Cleaning the nuclei
21. 
 

Prepare 5 mL as follows:
5 mL
0.02 % volume 
SECTION: Cleaning the nuclei
23. Centrifuge the flow-through 850 rcf, 5 min, 4 °C 
SECTION: Lysing the cells
15. Remove supernatant and add 250 µL

SECTION: Cleaning the nuclei
24. Remove supernatant and resuspend with 800 µL 
SECTION: Cleaning the nuclei
25. Centrifuge 850 rcf, 5 min, 4 °C 
SECTION: Cleaning the nuclei
26. Remove supernatant and resuspend in 500-1000 µL 

 

SECTION: Cleaning the nuclei
27. Centrifuge 850 rcf, 5 min, 4 °C 
SECTION: Cleaning the nuclei
22. Pass the sample through a 70 μm PluriStrainer filter to remove large chunks of undigested tissue.

 

SECTION: Cleaning the nuclei
28. Repeat wash steps 26 and 27: remove supernatant, resuspend in 500-1000 µL and centrifuge 850 rcf, 5 min, 4 °C 
SECTION: Cleaning the nuclei
29. Remove supernatant and resuspend in  500-1000 µL
Pass sample through a 40 μm PluriStrainer filter

 

SECTION: Using the nuclei
30. Count using Luna-FX7 or similar based on dual-fluorescence such as AO/PI. 
SECTION: Using the nuclei
31. 
 

SECTION: Using the nuclei
32. Rest on wet ice for immediate FACS cytometry analysis/sorting, or proceed to the Chromium X run using Chromium Fix RNA Profiling (10x Genomics) following the user guide for singleplexed or multiplexed samples accordingly. 
SECTION: Using the nuclei
32. Add 10 % (v/v) and 10 % (v/v) 
SECTION: Using the nuclei
33. Rest on ice for 10 min

SECTION: Using the nuclei
34. Cryopreserve at -80 °C 
SECTION: Using the nuclei
35. After storage, centrifuge 850 rcf, 5 min 

SECTION: Using the nuclei
36. Remove the supernatant without disturbing the pellet and continue from step 2.1a or even 2.1m (as recommended by 10x Genomics) of 2.1 Post-Hybridization Wash.